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A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants
Abstract
Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology.
... In the pursuit of deeper insights into R-proteins, researchers embarked on the cloning of resistance gene analogs (RGAs) using the degenerate primer technique [22][23][24][25]. This technique was applicable for the R-proteins with prior known conserved motifs (i.e. ...
... This technique was applicable for the R-proteins with prior known conserved motifs (i.e. P-loop to GLPL motifs) along with their degeneracy sequence information and it has been successfully applied in various plant species, such as rice, wheat, maize, barley, and vegetable crops [22][23][26][27]. While the degenerate primer technique has successfully facilitated the identi cation of RGAs, it also possesses several drawbacks such as reduced speci city, increased primer complexity, potential for non-target ampli cation due to the ambiguous nature of degenerate primers, limited coverage as they target conserved regions among related sequences, and the time-consuming process of primer design. ...
... While the degenerate primer technique has successfully facilitated the identi cation of RGAs, it also possesses several drawbacks such as reduced speci city, increased primer complexity, potential for non-target ampli cation due to the ambiguous nature of degenerate primers, limited coverage as they target conserved regions among related sequences, and the time-consuming process of primer design. Additionally, a common approach in developing degenerate primers for RGA identi cation involved comparing conserved motif sequences with reference species such as Arabidopsis (RPS2) and tobacco (N gene) [23,[28][29][30], trees [31][32][33][34]. These reference species have been extensively used to identify and characterize new RGAs in various crop species [22-23, 26, 28-42]. ...
Background: In the quest to identify new resistance genes analogous to those found in other plant species, a novel primer designing strategy is introduced for the first time. Unlike traditional methods that rely on prior information about degeneracy positions, this new approach involves designing primers based on specific domain positions within the candidate resistance gene and eliminates the need for prior knowledge of degeneracy. By using this new approach, it becomes possible to uncover resistance genes and understand their functional interactions with pathogens. Additionally, this approach sheds light on the redundancy and diversity of resistance genes. Notably, this primer designing strategy exhibits remarkable sensitivity, allowing the detection of elusive low-abundance target sequences that were previously challenging to identify using degeneracy-based designs.
Results: The qPCR primers, designed using the novel approach of protein domain-specific regions, underwent standardization and validation in endpoint PCR. Subsequent melt curve analysis in qPCR revealed that out of the ten primers tested, six NB-ARC family protein domain-specific qPCR primers (NB-ARC_2, NB-ARC_3, NB-ARC_4, NB-ARC_8, NB-ARC_12, and NB-ARC_17) exhibited a single peak melt curve, indicating precise amplification of the conserved NB-ARC domain of the R-protein. This confirms their specificity and reliability for target detection, enabling the identification of new resistance gene analogues.
Conclusion: Our innovative protein domain-specific qPCR primer design approach allows for precise and accurate PCR amplification, overcoming the limitations of traditional degenerate primers. It enables targeted amplification of specific domain regions within resistance proteins, uncovering both conserved domains and novel resistance genes or gene analogs. The use of these primers also captures the redundancy of resistance genes, offering improved accuracy and reliability in target gene identification. This breakthrough represents a significant advancement in molecular biology research and opens new possibilities for identifying resistance gene analogs. To the best of our knowledge, this is the first report of identifying resistance gene analogs using “protein domain-specific” region based qPCR primer design approach.
... NBS-LRR genes are characterized by highly conserved regions and these conserved domains in R genes offer opportunities to amplify and isolate analogues sequences from other plant taxa such as tomato sweet potato (Wang et al. 2010;Lozano-Torres et al. 2012). Various degenerate primers targeting these conserved domains have been designed and frequently used for the isolation of resistance gene analogues (Leister et al. 1996). ...
... Over the years, NBS-LRR protein is evidenced as major and significant multi gene family coupled in various plant disease resistance. The presence of conserved motifs within NBS-LRR class R genes was used to design degenerate primers for isolation, cloning of R gene family and for the understanding of R gene evolution (Leister et al. 1996;Dubey and Singh 2018). ...
... However, a detailed study is required by isolating more RGAs and analyzing their expression. Since RGAs incline to close proximity to disease resistance loci (Leister et al. 1996) they provide an ideal target for isolation of R genes and useful for breeding durable resistance. The PCR marker, developed in the present study can also provide a method to select resistant germplasm in chickpea breeding programme. ...
Management of chickpea wilt incited by Fusarium oxysporum f. sp. ciceris has been done primarily through development and use of resistant cultivars as part of an integrated management approach as the resistance genes are a major factor behind protecting plants from various pathogens. Thus, isolation and characterization of the RGAs of chickpea provide a critical foundation for deciphering host-pathogen interactions and development of novel methods to manage pathogens of crop plant. Eleven RGAs were identified in resistant variety (WR 315) of chickpea by degenerate PCR amplification followed by sequencing, cloning and database search. The restriction pattern generated using enzyme Hinf1 as well as analysis of phylogenetic tree of derived amino acid sequences classified chickpea RGA sequences into four classes of RGAs. Amino acid sequence alignment of these clones clearly showed the presence of consensus motifs namely, P-loop and Non-TIR linked sequences. The finding further supports that these consensus motifs are extensively present in dicot plant species. Three conserved motifs within the aligned sequences were also identified using MEME analysis. These conserve motifs exhibited the high level of similarity with NB-ARC domain as shown by pfam protein motif analyse. Quantitative real time-PCR analysis clearly revealed that both SA and JA induce the expression of RGA genes, however, the expression was higher in the plants treated with SA as compared to the control or JA treated plants. The differentially expressed signaling molecules suggesting that SA and JS as stimuli mainly involved in responding defence and activating signaling pathways.
... Actualmente han sido mapeados dos genes simples dominantes de resistencia extrema a PVY, el gen Ry adg derivado de S. tuberosum subsp. andígena (Leister et al. 1996) y el gen Ry sto derivado de S. stoloniferum (Brigneti et al. 1997) ...
... denominado Ry-f sto y está incorporado en las poblaciones de mejoramiento de países como Polonia Alemania y Holanda. Para su monitoreo se ha desarrollado el marcador CAPS GP122 718 que cosegrega con Ry-f sto y dista de dicho locus 1,2 cM (Flis et al 2004, aún sin publicar Leister et al. (1996) De los muchos RGLs obtenidos por Leister, tres fueron mapeados en el cromosoma XI en la región que contiene los genes Ry. Se diseñaron nuevos cebadores específicos para cada uno de ellos, a partir del clon diploide 2x(V-2)7 portador de Ry adg , se reamplificaron constatando que de estos tres, solo dos cosegregan con Ry adg , y de estos dos solo el RGL ADG2 es exclusivo de papa y no se amplifica en otras especies de solanaceas (Sorri et al. ...
... Figura 5. Comparación de la secuencia aminoacídica del fragmento ADG2 del parental resistente 2x(V-2)7 con la secuencias correspondientes a los genes N, RPP5 y RPS2. Las secuencias consenso entre N y RPS2 están subrayadas (P-loop; GGVGKTT y el dominio hidrofóbico GLPLAL), estas fueron empleadas por Leister (1996). Una la mutación en la posición Nº 260 del ADG2 es una mutación G C localizada dentro de un motivo kinasa-3a que resulta en una sustitución aminoacídica Ser Thr, las restantes son mutaciones silenciosas. ...
El virus Y de la papa (PVY) es el segundo patógeno viral mas importante a
nivel mundial y la principal causa de degeneración de la semilla en Uruguay.
Dos genes simples dominantes confieren resistencia extrema (ER) a PVY en
papa, el gen Ryadg derivado de S. tuberosum subsp. andígena y el gen Rysto
derivado de S. stoloniferum, siendo los mas empleados en los programas de
mejoramiento genético. Ambos se ubican en la misma región del cromosoma
XI, y junto con otros genes de resistencia integran una región rica en este tipo
de genes. La presencia de estos genes fue testada en 46 variedades y clones del
programa de mejoramiento de papa de INIA y de otros orígenes.
Para la detección de Ryadg se utilizó el marcador SCAR RYSC3 que fue
evidenciado en 19 genotipos, mientras que el empleo del marcador M45
permitió revelar la presencia de Rysto en un genotipo, contabilizando un total de
20 genotipos inmunes a PVY. Además, se comprobó que el marcador M45
resulta eficiente para testar la presencia de Ryadg y no solamente la de Rysto para
el cual había sido reportado por Brigneti et al. en 1997. Esta capacidad de
identificar simultáneamente ambas fuentes de resistencia extrema, lo destaca
como una herramienta eficiente y práctica para la selección de progenitores
resistentes al virus Y, y a diferencia de otras metodologías no requiere de
endonucleasas de restricción lo que permite reducir los costos y el tiempo de
estudio. Del trabajo realizado resulta relevante el alto porcentaje de genotipos
locales resistentes, evidenciando el interés prestado en el programa en
incorporar este tipo de resistencia en sus variedades, ya que el mismo confiere
una protección efectiva contra todas las cepas de PVY.
... Earlier genetic analyses demonstrated that 11 known R genes introgressed from Solanum demissum (Black et al., 1953). The clustering of functional genes for qualitative and quantitative resistance to various pathogens suggests their evolution from common ancestors by local gene duplication, followed by functional diversification (Gebhardt and Valkonen, 2001;Oberhagemann et al., 1999;Leister et al., 1996;Leonards-Schippers et al., 1994). ...
... Potato genotypes that have been developed and released before 2008 were from population type A . Other researchers also suggested that the clustering of functional genes for the resistance to various pathogens indicates they were from common ancestors (Gebhardt and Valkonen, 2001;Oberhagemann et al., 1999;Leister et al., 1996;Leonards-Schippers et al., 1994). Most of the varieties released before 2008 lost their resistance and exhibited genetic divergence from the recently released varieties. ...
Common bacterial blight (CBB) caused by Xanthomonas axonopodis pv. phaseoli is the most important biotic production constraints to common bean in eastern Ethiopia. Climate change could have an impact on the disease epidemiology of CBB by influencing both common bean growth and the pathogen reproduction. The effects of climate change needs to be mitigated using climate change resilience strategies. Field experiments were conducted in the 2012 and 2013 cropping seasons at Haramaya and Babile research stations in eastern Ethiopia to assess the effects of integrating climate change resilience strategies on CBB of common bean. These strategies used alone and in integration were compost application, row intercropping and furrow planting. Sixteen treatment combinations including sole planting (SP) were arranged in a randomized complete block design with three replications. Disease severity data were recorded from 10 randomly tagged plants from four central rows per plot. Disease severity, area under disease progress curve (AUDPC) and disease progress rate were significantly different among treatments, between varieties, cropping seasons and locations. Disease severities, AUDPC and disease progress rate were consistently less on row intercropping+ compost application+ furrow planting and row intercropping+ compost application than less integrated and SP plots in both locations and seasons. The disease epidemic was relatively higher on Mexican 142 than Gofta and during 2012 than 2013 at Babile than Haramaya. Integrated climate resilience strategies reduced CBB epidemics could be applied as a component
... Ritter, communication personnelle). Plusieurs homologues du gène N, conférant la résistance au Tobacco Mosaic Virus chez le tabac, ont été cartographiés dans ce cluster (Hamalainen et al. 1998, Leister et al. 1996. Le gène N fait partie de la famille des gènes de résistance de type TIR-NBS-LRR (Whitham et al. 1994). ...
... Des RGA ont été cartographiés chez de nombreuses espèces. Dans la plupart des études, des RGA se cartographient dans des régions génomiques où des gènes ou des QTL de résistance ont déjà été localisés (Chen et al. 1998, Geffroy et al. 1998, Hamalainen et al. 1998, Kanazin et al. 1996, Leister et al. 1996, Leister et al. 1998, Pan et al. 2000, Pflieger et al. 1999 Le chromosome XII de la pomme de terre est représenté dans la même orientation que sur les cartes génétiques des autres Solanacées. Cette orientation est inversée par rapport à celle observée en cytologie (Dong et al. 2000). ...
* INRA Centre d'Avignon, Documentation, Domaine St Paul, Site Agroparc, 84914 Avignon cedex 9 Diffusion du document : INRA Centre d'Avignon, Documentation, Domaine St Paul, Site Agroparc, 84914 Avignon cedex 9 Diplôme : Dr. d'Université
... PCR based resistance gene analogue (RGAs) approach may be a better approach in identification and cloning of resistance gene like sequences or putative candidate disease resistance gene or resistance gene analogues in sugarcane. Numbers of candidate disease resistance genes have been reported in many plants through this approach (Leister, et al., 1996;Gao, et al., 2010). Analysis of candidate disease resistance gene may enhance the understanding of biotic stress-responsive pathways in sugarcane and lead to the development of markers and diseases resistant cultivar for disease. ...
... The RGAs strategy has proven to be capable in identification and cloning of candidate disease resistance genes or RGAs related to biotic stress, molecular markers linked to disease resistance gene, and diversity analysis (Chen, et al., 1998). PCR based RGAs strategy has been successfully utilized for identification of putative disease resistance genes in various plant species (Leister, et al., 1996;Gao, et al., 2010;Nath, et al., 2013, Hameed, et al., 2015. Designing of primers utilized in identification of disease resistance genes based on data mining from various plant species (McIntyre, et al., 2005). ...
Sugarcane is an agro-economic crop, grown in tropical and subtropical regions of the world. Approximately 70% of world's sugar is produced by sugarcane. Sugarcane is also the major source of economically valuable products like ethanol and bio-electricity. India is the second largest producer of sugarcane crop in the world. Due to increasing world population, demand of the sugar is also increased. Quality and yield of sugarcane are extensively affected by pest, bacterial and fungal diseases. The urgent need of management of a given diseases is a requisite. Several resistance genes isolated from nucleotide binding site-leucine rich repeats (NBS-LRR) regions and kinase class of resistance genes are a valuable source to inculcate disease resistance in sugarcane and can be used for the development of resistant cultivars in future.
... PCR based resistance gene analogue (RGAs) approach may be a better approach in identification and cloning of resistance gene like sequences or putative candidate disease resistance gene or resistance gene analogues in sugarcane. Numbers of candidate disease resistance genes have been reported in many plants through this approach (Leister, et al., 1996;Gao, et al., 2010). Analysis of candidate disease resistance gene may enhance the understanding of biotic stress-responsive pathways in sugarcane and lead to the development of markers and diseases resistant cultivar for disease. ...
... The RGAs strategy has proven to be capable in identification and cloning of candidate disease resistance genes or RGAs related to biotic stress, molecular markers linked to disease resistance gene, and diversity analysis (Chen, et al., 1998). PCR based RGAs strategy has been successfully utilized for identification of putative disease resistance genes in various plant species (Leister, et al., 1996;Gao, et al., 2010;Nath, et al., 2013, Hameed, et al., 2015. Designing of primers utilized in identification of disease resistance genes based on data mining from various plant species (McIntyre, et al., 2005). ...
... This research leads in other legumes prompted us to hypothesize that homologs of AtTFL1 and AtLFY genes could also be present in horse gram and that it is possible to isolate and characterize them in horse gram. Our hypothesis is based on the rationale that sequence similarity and highly conserved functional domains and motifs of proteins encoded by TFL1 [phosphatidyl ethanolamine-binding proteins (PEBP) family] and LEAFY (LFY-family) genes even among distantly related crop species (PILLITTERI et al., 2004;KALDATE et al., 2021), offer opportunities to isolate and characterize homologs of TFL1 and LFY genes by PCR-amplification by degenerate primers designed to conserved motifs (LEISTER et al., 1996) in horse gram as well. To test the hypothesis, we attempted to isolate and characterize the horse gram homologs (MuTFL1 and MuLFY) of TFL1 and LFY genes using the reported degenerate primers designed for conserved motifs. ...
2023). Isolation and bioinformatics characterization of horse gram (Macrotyloma uniflorum (lam.) verdc.) homologs (MuTFL1 and MuLFY) of genes controlling growth habit.-Genetika, Vol 55, No.2, 491-503. Growth habit (indeterminate/determinate) is one of the evolutionarily shaped, economically important plant architectural traits in grain legumes, including horse gram. Arabidopsis AtTFL1 and AtLFY genes and their homologs in other grain legume species are known to control growth habit. Taking cues from highly conserved domains in protein encoded by TFL1 and LFY genes, we isolated horse gram homologs (MuTFL1 and MuLFY) genes using reported degenerate primers designed to conserved domains. MuTFL1 and MuLFY homologs were isolated and sequenced using Sanger's sequencing protocol. The nucleotide sequences of MuTFL1 and MuLFY homologs were translated to their corresponding amino acid sequences using "ExPASy" tool. BLASTx analysis of the translated amino acid sequences of MuTFL1 and MuLFY homologs showed high similarity with those of soybean and pigeon pea TFL1 and LFY homologs. Multiple sequence alignment of nucleotide sequences of MuTFL1 and MuLFY with those of related legumes and model species (Medicago sp. and Lotus japonicas) using "ClustalW" revealed the presence of four synonymous single nucleotide polymorphic (SNPs) sites in MuTFL1 and three non-synonymous SNP sites in MuLFY. These results suggest that not MuTFL1, but MuLFY could be the main regulator of growth habit in horse gram unlike in other non-model legumes where TFL1 was reported as the key gene controlling the growth habit. The substitution of glutamine in (determinate genotypes) with histidine 492 GENETIKA, Vol. 55 No2, 491-503, 2023 (indeterminate genotypes) in LFY-coded protein appeared to be the cause for switch over from indeterminate to determinate growth habit in horse gram. These results are discussed in relation to strategies for breeding horse gram cultivars with desired growth habits.
... In general, the RLK, TM-CC, and NBS-LRR genes account for a large proportion of R genes, playing important roles in plant disease resistance against fungal pathogens. Well-known examples include wheat leaf rust resistance conferred by the Lr21 (NBS-LRR) gene (Huang et al., 2003), resistance to the hemibiotrophic fungus Phytophthora infestans conferred by the potato R7 (NBS-LRR) gene (Leister et al., 1996;Hammond-Kosack and Jones, 1997), broad-spectrum mildew resistance conferred by the Arabidopsis RPW8 (TM-CC) gene (Xiao et al., 2001), and rice blast resistance conferred by the Pi-d2 (RLK) gene (Chen et al., 2006). The RLK, TM-CC, and NBS-LRR type RGAs associated with pasmo resistance in this study may contribute to a better understanding of the genetic mechanisms underlying pasmo resistance in flax. ...
Introduction
Flax (Linum usitatissimum L.) is an economically important crop due to its oil and fiber. However, it is prone to various diseases, including pasmo caused by the fungus Septoria linicola.
Methods
In this study, we conducted field evaluations of 445 flax accessions over a five-year period (2012–2016) to assess their resistance to pasmo A total of 246,035 single nucleotide polymorphisms (SNPs) were used for genetic analysis. Four statistical models, including the single-locus model GEMMA and the multi-locus models FarmCPU, mrMLM, and 3VmrMLM, were assessed to identify quantitative trait nucleotides (QTNs) associated with pasmo resistance.
Results
We identified 372 significant QTNs or 132 tag QTNs associated with pasmo resistance from five pasmo resistance datasets (PAS2012–PAS2016 and the 5-year average, namely PASmean) and three genotypic datasets (the all SNPs/ALL, the gene-based SNPs/GB and the RGA-based SNPs/RGAB). The tag QTNs had R² values of 0.66–16.98% from the ALL SNP dataset, 0.68–20.54%from the GB SNP dataset, and 0.52–22.42% from the RGAB SNP dataset. Of these tag QTNs, 93 were novel. Additionally, 37 resistance gene analogs (RGAs)co-localizing with 39 tag QTNs were considered as potential candidates for controlling pasmo resistance in flax and 50 QTN-by-environment interactions(QEIs) were identified to account for genes by environmental interactions. Nine RGAs were predicted as candidate genes for ten QEIs.
Discussion
Our results suggest that pasmo resistance in flax is polygenic and potentially influenced by environmental factors. The identified QTNs provide potential targets for improving pasmo resistance in flax breeding programs. This study sheds light on the genetic basis of pasmo resistance and highlights the importance of considering both genetic and environmental factors in breeding programs for flax.
... Sequence analysis of LaRGAP63 showed 85.40 and 84.29 per cent homology with mosaic virus resistance protein and disease-resistant protein RPP-4, respectively. A number of RGAs were found to be linked with virus, bacteria and nematodes resistance in many crop species (Kanazin et al., 1996;Leister et al., 1996;Yu et al., 1996;Speulman et al., 1998;Spielmeyer et al., 2000). Saha et al. (2013) also identified one molecular marker (sgRGC 18) linked with ToLCNDV resistance in sponge gourd. ...
Yellow mosaic disease caused by Tomato leaf curl New Delhi virus (ToLCNDV) causes 100 percent losses in ridge gourd under epidemic conditions, particularly in the tropics and sub-tropics of India. Plant breeding approaches led by the marker-assisted selection have gained increased momentum in virus resistance breeding to hasten the development of resistant varieties. In the present study, an effort has been made to identify molecular markers linked to yellow mosaic disease resistance loci in an F2 population derived from a cross between susceptible ‘Arka Prasan’ and resistant ‘IIHR-Sel-1’ of ridge gourd. All the molecular markers were amplified in parents, and one polymorphic marker clearly distinguished the contrasting parents. The primers LaRGAP 63 produced a polymorphic DNA fragment that co-segregated with yellow mosaic disease reaction phenotypically in the F2 population. The identified marker will be helpful to the breeders for introgression of resistance loci into the elite background.
... Majority of R genes encode for the NBS-LRR protein class, with a characteristic nucleotide-binding site domain (NBS) and a leucine-rich domain (LRR). A PCR-based strategy has been proved to be an efficient approach for the isolation of R genes or resistant gene analogues (RGAs) in several crops such as potato [12] soybean [13] and maize [14]. ...
This study was carried out to investigate the phenotypic reaction of 21 selected chilli accessions available in Sri Lanka for chilli anthracnose caused by mixed infection of Colletotrichum capsici and Colletotrichum gloeosporioides and protein level changes underlying with the plant response. A mixed inoculum of 10 5 conidia ml-1 of fungus was used and disease severity index (DSI) were calculated. A set of primers, designed based on resistant gene analogues (RGA) was used for PCR amplification of selected possible resistant gene sequences. Amino acid profiles were generated and were compared with similar amino acid profiles available in the NCBI. The results revealed that chilli accessions show varying degrees of susceptibility from low to high for anthracnose and none were resistant to anthracnose. The lowest DSI values reported from MI Hot (19.47), Hen miris (24.73), Galkiriyagama selection (26.74), C. baccatum (26.95) and MICH3 (27.03). The amino acid profiles showed that RGAs of chilli accessions, except MICH3 belonged to NB-ARC domain. Homology analysis on amino acid profiles showed a high diversity among the studied RGAs and varying susceptible reactions observed among 21 chilli accessions to the anthracnose disease caused by C. capcisi and C. gloeosporioides may be due to non-formation of required proteins as a result. MI Hot, Hen Miris, Galkiriyagama selection, C. baccatum and MICH3, which showed lower susceptibility to anthracnose disease, can be used in different combinations in breeding Original Research Article Kumari et al.; ARJOCS, 4(1): 111-124, 2022; Article no.ARJOCS.729 112 programmes to improve anthracnose resistance in chilli. Molecular and protein level findings of this study may be useful in creating genetic resistance for anthracnose caused by C. capsici and C. gloeosporioides.
... Among the most reproducible QTLs were Eca1A on chromosome one, which may be linked to the self-incompatibility locus and Eca6A on chromosome two. These two regions are anchored to genomic regions in tomatoes that house resistance genes to a variety of pathogens (Leister et al., 1996). QTL analysis solidified the likelihood that PBSR resistance is a polygenic trait which follows a complex pattern of inheritance (Zimnoch-Guzowska et al., 2000). ...
Solanum tuberosum L. is the world's most important non-cereal food crop, capable of producing more food per land unit on less water than any other crop. Only rice, wheat, and maize are produced in larger quantities than potato. The potato tuber, a modified stem turned storage organ is nutrient dense and a staple in diets across the world. The potato crop is expected to grow and contribute significantly to the global food supply. However, potato production has increasingly been threatened by unfavorable environmental conditions, and susceptibility to pest and disease. Perhaps the most famous of all the Irish Potato Famine caused by the oomycete Phytophthora infestans. Constraints on potato production through abiotic and biotic factors have been tackled through crop improvement or breeding programs. As a tetraploid crop, the potato breeding cycle can take many years from conception to variety release. This process takes on average,10 years to complete. This lengthy breeding cycle does not mean that traditional potato breeding is unsuccessful, simply resource intensive. Due to the demands on available resources caused by potato breeding cycles, some pest and pathogen resistances may not be incorporated into commercial germplasm, which is further influenced by the amount of pressure the pathogen exerts annually on the potato crop. The tetraploid nature of the crop and the ease of which tubers can be obtained and shipped has led to a vegetative propagation system for seed tubers. Although regulated through certification programs, diseases are still able to penetrate these proactive cultural practices as was the case in, 2015 when there was an outbreak of Dickeya dianthicola struck the Northeast potato growing regions in the U.S. and quickly spread throughout the country because of limited control measures. A recently released variety from the University of Maine Breeding Program, Caribou Russet, was shown to have tolerance to potato blackleg and soft rot (PBSR) caused by Dickeya dianthicola strain ME30. In this work, an effective Dickeya dianthicola isolate ME30 culturing method and inoculation workflow is established to repeatedly and reliably phenotype for the potato blackleg soft rot resistance phenotype. Using this workflow and a population of primary dihaploid Caribou Russet, the underlying genetic source of tolerance observed at the tetraploid state is sought. This work provides a workflow to enable reliable means of phenotyping PBSR resistance in Caribou Russet, as well as incorporating and identifying PBSR resistance into future germplasm for potato breeding. Potato haploid induction holds promise in revitalizing the industry through implementation of a true potato seed system via a diploid potato breeding system. Potato haploid induction also allows researchers to investigate the genetics of agronomically important traits in a setting with less complex epistatic effects, and simpler segregation ratios. By leveraging haploid induction crosses in the cultivated potato, the underlying genetic sources of tolerance to PBSR infection can be mined from the potato genome. Dickeya dianthicola isolate ME30 characterization resulted in the creation of a linear regression model which describes the relationship of optical density at 600nm to the estimated colony forming units per milliliter of culture, as well as the identification of an optimal inoculum concentration at which to vacuum infiltrate tubers for disease phenotyping. Subsequent validation of this workflow occurred during the disease phenotyping experiment on a population of primary dihaploid Caribou Russet. These data were then used for QTL mapping. QTL mapping revealed no significant QTL. Nonetheless, a peak was detected along chromosome 6 aligning with previous published literature on PBSR resistance. Thus, suggesting that there is an underlying genetic source of resistance within the primary dihaploid Caribou Russet gene pool.
... Nevertheless for quantitative traits also, when biological functions of the gene or genes are directly associated with the trait of interest, CG approach could be considered as a viable alternative strategy for effective marker-assisted selection (MAS) (Pflieger et al. 2001). The CG approach has been used widely for QTL identification and MAS with success in several crop plants like maize Zea mays L. (Byrne et al. 1996), tomatoes Solanum lycopersicum L. (Boylan and Quail 1991), pepper Piper nigrum (Lefebvre et al. 1998), potato Solanum tuberosum L. (Leister et al. 1996), etc. In tree crops, CG approach was used to identify markers from drought-adaptation and photosynthesis-related gene loci in poplar (Chu et al. 2009), lignin composition in Eucalyptus (Mandrou et al. 2011), disease resistance in apricot (Mariette et al. 2016), etc. Natural rubber is a high molecular weight polymer whose structure is cis-1,4-polyisoprene. ...
Scarcity of functional genetic markers associated with candidate genes (CGs) is a serious constraint for marker-assisted selection in the natural rubber producing tree, Hevea brasiliensis. In order to develop markers associated with rubber yield, five CGs involved in latex biosynthesis were characterized from 16 popular Hevea varieties. Novel SNPs and indels were identified and developed into markers using simple genotyping techniques like allele-specific PCR, CAPS, etc. A progeny population was genotyped using these markers to validate them, to understand their segregation pattern and to map them to a genetic linkage map. Parent-specific maps were constructed using pseudo-test cross strategy with the help of additional markers. The sequence structure information generated will be useful for future studies on gene mapping, functional relevance of coding SNPs and evolution of rubber biosynthesis genes in Hevea. Concurrently, the markers developed may serve as powerful tools for yield-based selection and for genetic diversity and pedigree studies in Hevea. Above all, the marker assays designed for genotyping could be economically carried out in any laboratory having basic molecular biology infrastructure and expertise.
... To understand processes like these, scientists need access to a diverse and growing variety of Cannabis plants which are not available through NIDA. Important discoveries in other plant groups, such as transposable elements (McClintock, 1950), genes related to pathogen resistance (Leister et al., 1996), or genes related to yield (Sakamoto and Matsuoka, 2008) would have not been possible had there been similar restrictions on their research. This limitation also affects the untapped possibilities of using Cannabis to treat a multitude of illnesses, with enough anecdotal evidence from consumers to merit rigorous scientific investigation, using strains that are reflective of those used by consumers claiming medicinal and/or therapeutic effects. ...
The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genomes of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDA’s varieties differ from widely available medical, recreational, or industrial Cannabis. Furthermore, our results suggest that NIDA’s varieties lack diversity in the single-copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome. Therefore, results based on NIDA’s varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation.
... New primers can easily be incorporated into this PCR based assay, thereby providing a means for continually updating and improving the specific and sensitivity of the assay. Degenerate Primers are also used to direct the amplification of specific plant rice, barley, maize, sugar beet, wild apple etc (Hunger et al. 2003, Leister et al. 1996and Pan et al. 2000a). So amplification of eggplants DNA through RT-PCR by using degenerate primer was also positive. ...
... Previous researchers suggested that the clustering of functional genes for the resistance to various pathogens argues the genotypes have come through common ancestors (Gebhardt and Valkonen, 2001;Leister et al., 1996;Valério et al., 2005;Crouch et al., 2006). The two old released varieties (Seredo and Gambella) lost their resistance to anthracnose and demonstrated genetic divergence from the recently released varieties (Arghiti and Dekeba) even with the old variety (76TI#23). ...
Anthracnose disease caused by Colletotrichum sublineolum is a major constraint that limits the production and productivity of sorghum in Ethiopia as well as in other countries. This study was carried out at Arba Minch and Derashe districts during the 2018 cropping season, March to August. The objective of the study was to identify the host resistance for anthracnose management and estimate the genetic variability in terms of resistance to anthracnose, growth, and yield potentials of sorghum. The treatments used in the study were 14 sorghum genotypes and laid in a randomized complete block design with three replications. The results from this study showed that Arghiti, Dekeba, and 76TI#23 were found resistant, while Kolamulato-W, Konso-R, Gambella, and Seredo were highly susceptible to anthracnose. The lowest (0.25 t ha-1) grain yield was recorded from all landraces, whereas the highest (3.22 t ha-1) grain yield was obtained from the genotype Melkam. The high phenotypic and genotypic variances were figured out for the study parameters, while the environmental variance was lower than the genotypic and phenotypic variances. Low to high genotypic (21.40 to 57.94%) and phenotypic (22.66 to 95.21%) coefficients of variations were computed for the study parameters. The high heritability of 59.30 to 99.63% and the genetic advance in percent means of 20.21 to 63.10% were calculated for the study parameters. The disease parameters, like disease severity and area under disease progress curve (AUDPC) (rg = 0.725), showed a strong and positive association of genotypic coefficients correlations. Also, strong and negative genotypic coefficients of correlations were observed between the disease and traits studied, like AUDPC and grain yield (rg = -0.561). The dendrogram of the sorghum genotypes using the Unweighted Pair-Group Method with Arithmetic means analysis and Euclidean distances separated the genotypes into three clusters. The result from this study revealed that the sorghum genotypes noticeably varied in resistance to anthracnose as well as growth and yield potential. Therefore, sorghum producers could beneficially produce resistant, moderately resistant, and high-yielding genotypes with minimum integrated management efforts against the anthracnose. Moreover, the genetic variability and the high heritability together with, and the high genetic gain of the characters, suggest the potential of bettering the genotype for disease resistance and high yield potential through selection.
... The conserved motifs within plant NBS domains make it possible to design degenerate RGAs primers for PCR based amplification. Several disease resistant gene homologues have been cloned in monocot and dicot species (Leister et al., 1996;Kanazin et al., 1996;Yu and Maroof, 1996). ...
Mungbean (Vigna radiata (L.) Wilczek) (2n=22) is an
excellent source of easily digestible proteins with low flatulence
which complements the staple rice diet in Asia. The estimated
genome size of mung bean is 579 Mb. The average yield of
mungbean is very low, not only in India, but also in e ntire
tropical and subtropical Asia. Among the various viral diseases,
Yellow Mosaic Virus is most destructive viral disease and l eads
to severe yield reduction. Conventional breeding for
incorporation of disease resistance is time consuming and less
efficient as compared to marker assisted selection (MAS).
In order to identify the marker linked to yellow mosaic
virus resistance gene, MYMV resistant genotype Meha and �MYMV susceptible high yielding variety GM-4 were crossed and
subsequently F5 RIL population was developed through single
seed descent method. Both parents and 165 F5 RILs were
subjected to natural screening to assess their resistance or
susceptibility against MYMV infection. This study revealed that
disease resistant reaction was inherited as a monogenic trait. F5
mapping population segregated in 85 resistant : 80 susceptible
RILs in accordance with expected ratio 1:1 which was confirmed
by chi-square analysis showing that MYMV resistance is under
monogenic control.
A total of 124 primers used to survey parental
polymorphism between parents, only one RGA primer pair 1F�CG/RGA 1R was able to deduce polymorphism with an allele
size 445 bp. The RGA primer pair amplified this fragment in
resistant parent ‘Meha’, resistant bulk and 10 resistant RILs,
while it was absent in susceptible parent ‘GM-4’, susceptible
bulk and 10 susceptible RILs. The amplification of resistant
parental allele in resistant bulk and not in susceptible parent and
susceptible bulk indicated that this marker is associated with the
gene controlling MYMV resistance in green gram.
Significant chi-square estimate of 69.69 (p > 0.05) for co�segregation between disease resistant gene and RGA marker
assuming independent assortment ratio of 1:1:1:1 in F5 RILs of
mungbean revealed presence of linkage. The result of linkage
analysis for RGA marker exhibited location at the distance of� 18.35 ± 0.029 cM from locus governing disease resistant
reaction. The results are also in accordance with the lineage of
mosaic resistance in Meha from blackgram.
The information available from present study may aid to
impart mosaic resistance in high yielding but susceptible
varieties using marker assisted selection. High resolution
mapping of gene(s) involved in MYMV resistance may facilitate
marker assisted introgression of disease resistant gene in high
yielding susceptible genotypes.
Development of codominant marker would increase
efficiency of marker assisted selection even in early segregating
generations. Sequencing of the RGA marker obtained in present
study and homology search with mungbean / pulse genome
database may provide more insights about functiona l role of this
marker, if any, in imparting MYMV resistance.
Utilization of next generation sequencing based genomics
as well as transcriptomics tools and mapping population like
recombinant inbred lines (RILs), near isogenic lines (NILs) and
residual heterozygous line (RHL) may further accelerate the
process of molecular dissection of MYMV resistance.
... Prior to the development of next generation sequencing technologies, the isolation of subsets of plant NLRs across different plant species was achieved using degenerate primers for targeting known conserved domain motifs previously identified in model species, such as the NBS Ploop, GLPL and RNBS motifs (Kanazin, Marek, & Shoemaker, 1996;Leister et al., 1998). Considerable work on PCR cloning of target Resistance Gene Analogs (RGAs) has been reported during the last 25 years across a wide range of dicot species, including Arabidopsis thaliana (Aarts et al., 1998;Meyers et al., 1999;Speulman, Bouchez, Holub, & Beynon, 1998), cotton (He et al., 2004), soybean (Peñuela, Danesh, & Young, 2002), peanut (Bertioli et al., 2003), potato (Leister, Ballvora, Salamini, & Gebhardt, 1996), and perennials such as apple (Baldi et al., 2004). For a review of the extension of this approach see Sharma, Das, Kumar, & Lodha, 2009. ...
... The RGA markers have conserved regions and can represent sites of biological relevance in the phenotypic expression of resistance genes, thus presenting themselves as a useful tool for different genetic approaches (Bent, 1996). Based on phylogenetic conservation, different primers were developed in order to amplify regions analogous to resistance genes (Leister et al. 1996, Kanazin et al. 1996, Yu et al. 1996. Thus, markers of this nature make it possible to obtain a lot of polymorphism between different accessions of the same species or different species of genus, which may corroborate in systems of genetic characterization of accessions and cultivars (Hammond-Kosack & Jones, 1997). ...
Brazil is highlighted as the largest producer and consumer of passion fruit (Passiflora) and holder of representative biodiversity. Studies dedicated to the genetic characterization of the genus in order to understand the existing diversity are important for the advancement of genetic improvement and conservation programs. The aim of this study was to select combinations of RGA (Resistance Genes Analogs) primers for commercial passion fruit species and varieties. To this end, amplification tests were performed on a PCR platform in 17 combinations of RGA primers for eight species and 12 commercial varieties of passion fruit from the Banco Ativo de Germoplasma Flor da Paixão. All combinations of primers have generated amplification products in at least one of the Passiflora samples. The number of combinations that generated amplification products varied from seven to 12 in the species and from three to eight in the varieties. Three to 12 combinations generated amplification products for the samples, emphasizing that the RGA markers were efficient in accessing genomic loci in the species and varieties of passion fruit. The variation of efficient combinations in each of the species and varieties attested to the importance of this research in the preliminary stages of genetic studies as auxiliary tools in conservation and genetic improvement programs. However, this study made it possible to identify combinations of primers to be prioritized in inter and intraspecific genetic characterizations of passion fruit.
... The majority of the R gene analogs (RGA) belong to NBS-LRR superfamily and the presence of such structural motifs makes them potential regions for providing disease resistance (Li et al. 2016). Earlier, the technique for identification of resistance gene analogs capitalizes on conserved regions for designing degenerate primers and isolation using the polymerase chain reaction (PCR) (Leister et al. 1996;Kanazin et al. 1996). Genome-wide investigations revealed that RGAs accounts for approximately 0.2-1.3% of genes predicted in plant genomes where they occur in clusters, and however RGA content varies with different plant genotypes (Ameline-Torregrosa et al. 2008;Yang et al. 2008;Bayer et al. 2019). ...
Black pepper (Piper nigrum L.) is an important spice crop with high economic value. However, its production is severely hampered by the oomycete pathogen, Phytophthora capsici. Integrative disease management strategies have been developed to control the pathogen, but the pathogen is in the phase of evolving its virulence. Absolute resistance against Phytophthora rot was not reported in black pepper germplasm. However, Piper colubrinum, a wild species is reported as resistant. Resistance proteins are involved in continuous surveillance of pathogen entry and activation of plant defense signalling pathways for an effective hypersensitive response to prevent pathogen invasion. In this study, a sequence-based homology approach using the conserved nucleotide-binding site (NBS) of known plant resistance genes was used to isolate Resistance Gene Analogs (RGA) and assess their transcript level during Phytophthora infection. The RGA transcript level was evaluated in resistant wild species (P. colubrinum), two moderately resistant black pepper genotypes (IISR Sakthi and 04-P24-1), and one susceptible genotype (Subhakara). The identified RGAs of black pepper were found to be of non-TIR R gene class with NBS motifs. The expressions of six PnRGAs were assessed employing qRT-PCR at different time points after challenging with highly aggressive Phytophthora isolate. The kinetics of differential expression post-infection with P. capsici indicates the differential timing and magnitude of pathogen recognition in resistant P. colubrinum and moderately resistant black pepper genotypes compared to susceptible genotype. In silico analysis revealed that differentially expressed P. nigrum RGAs function through ADP phosphorylation, which is a key process in pathogen recognition. The identification of P. nigrum RGAs induced by P. capsici should provide valuable information for cloning and characterization of resistance genes.
... Prior to the development of next generation sequencing technologies, the isolation of subsets of plant NLRs across different plant species was achieved using degenerate primers for targeting known conserved domain motifs previously identified in model species, such as the NBS Ploop, GLPL and RNBS motifs (Kanazin, Marek, & Shoemaker, 1996;Leister et al., 1998). Considerable work on PCR cloning of target Resistance Gene Analogs (RGAs) has been reported during the last 25 years across a wide range of dicot species, including Arabidopsis thaliana (Aarts et al., 1998;Meyers et al., 1999;Speulman, Bouchez, Holub, & Beynon, 1998), cotton (He et al., 2004), soybean (Peñuela, Danesh, & Young, 2002), peanut (Bertioli et al., 2003), potato (Leister, Ballvora, Salamini, & Gebhardt, 1996), and perennials such as apple (Baldi et al., 2004). For a review of the extension of this approach see Sharma, Das, Kumar, & Lodha, 2009. ...
In order to meet global food security demands in the next decades, considerable changes are required for sustainable agriculture in the context of plant disease, with sufficient food production depending on the development of durable genetically disease resistant crops. For this, further advances are required in our understanding of the plant innate immune system and how plants respond to invading pathogenic micro-organisms. Over the past 20 years, considerable research has been conducted into the characterization and cloning of plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors. These intracellular receptors can recognize directly or indirectly pathogen effector proteins, resulting in effector-triggered immunity (ETI). Elucidation, however, of the diversity of NLR resistance gene families and the molecular basis of NLR-driven effector recognition and defense signaling is incomplete. Here, we present a summary of the understanding of NLR structure, function, genomic organization and diversity in plants. Recent advances in target enrichment approaches for NLR characterization and function validation are highlighted in the context of NLR engineering possibilities for accelerated durable genetic resistance to biotic stresses.
... Among the isolated R genes, 5 classes are identified based on common characteristics that include the nucleotide binding site, leucine-rich repeats, a transmembrane domain and a serine/threonine protein kinase domain (Bent, 1996;Ellis and Jones, 1998;Hammond-Kosack and Jones, 1997). Genetic mapping of resistance gene specificities has indicated that they frequently cluster at complex loci and cosegregate with different resistance specificities ( Jia ef al., 1997;Leister et al., 1996;Ori etal., 1997;Pan etal., 2000;Parniske etal., 1997;Song etal., 1997). The shared motifs among R proteins identified in different plant species and for different pathogen types, suggest conservation in controlling strain-specific pathogen resistance in plants by similar signalling mechanisms. ...
The late blight disease, caused by the oomycete Phytophthora infestans (Mont.) de Bary, is a serious threat to the potato crop every growing season. This has, for example, led to the disastrous Irish famine in the middle of the 19 th century, and continued in the 20 th century to remain a serious problem for potato growers. Since the early 1980s P. infestans populations changed more rapidly and epidemics even increased in their severity. Resistance breeding stimulated the introduction of resistance genes ( R genes) from wild Solanum species into cultivated potato, Solanum tuberosum , but newly occurring virulent races of P. infestans circumvented these R gene mediated resistances and no cultivars with durable resistance were obtained. At the moment, methods using fungicides supervised by spraying control via decision support systems are the only available control measures.
Characteristic for R gene type mediated resistance reactions is the hypersensitive response (HR) leading to local cell death causing necrotic spots at the site of attempted infection. Genetic analysis of HR mediated resistances showed that activation of HR is highly specific and induced upon recognition between a specific R gene in the plant and a corresponding avirulence gene ( Avr gene) in the pathogen. Insights in the molecular mechanisms underlying this HR resistance reaction in Solanum species might facilitate the development of potato cultivars that are more durable in maintaining a resistance phenotype.
A two component Ac-Ds transposon tagging strategy in diploidised potato was developed to identify and isolate genes involved in the R1 gene mediated resistance response to P. infestans . Transposable elements are molecular genetic tools to mutate and identify genes. The transposable elements Ac and Ds were first characterised in maize and their molecular isolation led to the identification of maize genes that were tagged by these elements. The autonomous Ac element is able to transpose by itself and also to induce transposition of the non-autonomous Ds element that is transposase defective. Introduction of these elements in heterologous species demonstrated their utility for isolating genes in self-fertilising plant species. Also in the highly heterozygous and tetraploid potato, the Ac and Ds transposable elements were shown to be functional. A cell autonomous visual marker gene for potato, the granule bound starch synthase gene ( GBSS gene), enabled a refined characterisation of Ac transposition in potato. Further molecular characterisation showed high levels of Ac-Ds transposition both somatically and germinally, so that suitable populations could be generated for tagging purposes.
The production of clones homozygous for the gene of interest that are normally required for efficient tagging strategies, turned out to be time consuming in potato due to self-incompatibility at the diploid level. Therefore, an alternative method based on somatic transposition was developed for the direct selection of transposition events instead of recovering germinally transmitted transpositions. Highly chimaeric Ac-Ds seedlings with active Ds transposition linked to the R1 resistance gene on chromosome 5 of potato were selected. Protoplasts were isolated from actively transposing seedlings and using the hygromycin excision selection marker, regenerants could be selected with new independent Ds insertions. The resulting R1 resistant transposon mutagenised population of almost 2000 hygromycin resistant regenerants formed an ideal start for the identification of an R1 tagged mutant, or other Ds insertional mutants with an altered R1 resistance response.
The somatically regenerated tagging population was analysed for the P. infestansR1 type HR resistance response, using a detached leaf assay for P.infestans inoculation. In a primary screening, 33 potential R1 resistance variants showing partial susceptibility to P. infestans race 0 were identified. These results were further quantified using stringent inoculation conditions on replicate samples leading to the identification of four putative mutants with a distinctly altered R1 resistance response. In these putative mutants less than 50% of the inoculated leaves showed the R1 type HR response and clear colonisation with sporulation of P. infestans was observed. The flanking sequences of the Ds insertion sites in these putative R1 mutants were analysed and in two cases a potential biological correlation between the insertion sequences and the phenotype was evident. One putative mutant contained a Ds insertion in a region with auxin and abscisic acid response cis-elements homologous to a specific region (TAPIR) of the tomato defence related genes TAPG2 and TAP1 .
The second P. infestansR1 resistance mutant, mutant 1000 with a striking susceptible phenotype was characterised in more detail. Two Ds insertions were identified and the insertion site flanking sequences both showed high homology to serine/threonine protein kinases. The Ds insertion sites turned out to be homologous but not identical, indicating two independent Ds insertions in homologous but not identical genes. Both sequences showed protein identity to all the conserved regions of serine/threonine protein kinases and they contained a conserved intron position. The closest homology was to the serine/threonine protein kinase domain of the Xanthomonas resistance gene Xa21 , which is involved in the induction of a HR resistance response in rice. This indicates that the isolated Solanum tuberosum protein kinase (StPK) homologs are candidate genes involved in resistance gene activity in potato. Further specific molecular analyses identified at least 11 homologs by sequence, which probably belong to a large family of serine/threonine protein kinases in potato. Both homologs in which the Ds transposons are inserted were present in susceptible parental potato clones. Therefore, it is unlikely that the isolated sequences represent the R1 gene itself. The mutated StPKs were designated rpr1 and rpr2 , r equired for P hytophthora infestansr esistance gene 1 and 2. Studying these mutants and the StPKs involved might help in understanding the pathway leading to HR resistance in potato.
... The NBS-R3 gene could be a candidate for resistance to downymildew in sunflower. The same approach was used in recent studies to map candidate disease-resistance genes in soybean (Kanazin et al 1996, Yu et al 1996 and in potato (Leister et al 1996). ...
... آنالوگ تنوع ژن های نان گندم زنگ به حساس و مقاوم ارقام در بیماری به مقاومت های Leister et al., 1996;Ameline-Torregrosa et al., 2008;Arya et al., 2014;Sekhwal et al., 2015;Kassa et al., 2017 .) توالی ...
Fungal diseases, especially leaf and stripe rusts are wheat yield reducing factors in Iran and the world. In this study, genetic diversity of 20 wheat varieties with different response to leaf and stripe rusts were studied using primers designed based on the conserved regions of plant disease resistance genes. The banding patterns of polymorphic markers were scored as dominant and number of amplified bands and percentage of polymorphism were determined. In addition, for each primer combination, polymorphic information content (PIC) and marker index (MI) were calculated. Out of the 11 used single primers and primer combinations, five primer combinations and a single primer produced scorable amplification. The maximum and minimum of PIC were observed for LLOOP-1 and H2016-H2020 primer combination with mean value of 0.50 and 0.28, respectively. The primer combinations of H2016-H1146 and H2016-H2020 with mean values of 4.80 and of 2.84, had minimum and maximum of MI, respectively. Cluster analysis based on Neighbor-Joining algorithm and evolutionary P-distance coefficient assigned the varieties into four groups which were in agreement with their response to yellow rust. In principal coordinate analysis, the scatter plot of varieties based on two first coordinates confirmed the groups obtained from cluster analysis.
... Using the sequence motifs of resistance genes isolated from tobacco and Arabidopsis, Leister et al. (1996) were able to isolate potato resistance genes against nematode infection. One of these genes was linked to the nematode resistance loci Gro1 and R7 responsible for resistance against Phytophthora infestans. ...
Plant-parasitic nematodes cause major agricultural losses worldwide. Examining the molecular mechanisms underlying plant-nematode interactions and how plants respond to different invading pathogens is attracting major attention to reduce the expanding gap between agricultural production and the needs of the growing world population. This review summarizes the most recent developments in plant-nematode interactions and the diverse approaches used to improve plant resistance against root knot nematode (RKN). We will emphasize the recent rapid advances in genome sequencing technologies, small interfering RNA techniques (RNAi) and tar- geted genome editing which are contributing to the significant progress in understanding the plant-nematode interaction mechanisms. Also, molecular approaches to improve plant resistance against nematodes are con- sidered.
... This feature has been widely exploited to identify similar sequences in other plant species using PCR. These sequences are generically known as resistance gene analogs (RGAs) ( [12][13][14][15] and references cited therein). When primers targeting conserved domain sequences -such as the NBS (Nucleotide Binding Site) domain of several R genes -are used in combination with polyacrylamide gels, the number of RGAs detectable can be increased. ...
Background
Protein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the reliable identification of the full range of genes involved. The present work reports the technique of protein kinase (PK)-profiling for the identification of the PK genes induced in the partially resistant oats line MN841801–1 following exposure to the fungus Puccinia coronata. This is the first time this technique has been used with cDNA (complementary DNA) from a suppression subtractive hybridization library obtained after the hybridization of cDNAs from inoculated and mock-inoculated plants.
Results
Six degenerate primers based on the conserved domains of protein kinases were used in a PK-profiling assay including cDNA from mock-inoculated leaves and subtracted cDNA. Of the 75.7% of sequences cloned and sequenced that showed significant similarity to resistance genes, 76% were found to code for PKs. Translation and ClustalW2 alignment of each sequence cloned with the complete sequences of the most similar B. distachyon PKs allowed those of the partially resistant oat line to be deduced and characterized. Further, a phylogenetic study carried out after alignment of these B. distachyon PK sequences with the most similar protein sequences of related species also allowed to deduce different functions for the PK cloned. RT-qPCR (Reverse Transcription-quantitative PCR) was analyzed on nine representative sequences to validate the reliability of the employed PK-profiling method as a tool for identifying the expression of resistance-associated genes.
Conclusions
PK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata, and perhaps other pathogens. Most of the PKs studied are related to receptor-like protein kinases expressed shortly after infection. This is in agreement with previous studies indicating a close relationship between partial resistance and the first layer of defense against pathogen used by plants.
Electronic supplementary material
The online version of this article (10.1186/s12870-018-1604-y) contains supplementary material, which is available to authorized users.
... The most important form of control is the selection of resistant genes by plant breeders (Ballvora et al., 2002;Leister et al., 1996); however; this provides a direct selective pressure through which pathogens can overcome the resistance (Fry & Goodwin, 1997). Therefore, where single gene (qualitative) resistance is adopted, an arms race exists between plant breeders and the pathogen which is not sustainable in the long run. ...
Phytophthora species cause many major plant disease epidemics worldwide,
impacting horticulture, plant trade and production planting industries through the
loss of plants and the costs arising from disease management. Phytophthora
species have specialised structures which enhance their dispersal, survival and
infection. These include motile swimming spores, caducous sporangia and resting
spores. Nurseries and orchards use integrated management to control
phytophthora diseases, including cultural practises, biological control, resistant
host plants and chemical control. The use of fungicides is a very effective control
method; however, Phytophthora species can develop resistance to fungicides after
prolonged exposure. For example, phosphite tolerant isolates of Phytophthora
cinnamomi have been found in avocado orchards in Australia and South Africa.
Phosphite is widely used in nurseries and orchards in New Zealand as a
preventative and control treatment. The avocado industry largely relies on it for
the control of avocado root rot caused by P. cinnamomi. Phosphite is most
commonly applied to avocado trees using passive trunk injections but can also be
applied as foliar sprays, soil drenches, high-pressure injections and capsule
implants.
To assess phosphite tolerance of P. cinnamomi isolates from New Zealand
avocado orchards, a high-throughput optical density (OD) assay was developed to
measure mycelial growth inhibition in the presence of phosphite. The OD assay
was used to screen 24 P. cinnamomi isolates from four orchards never treated with
phosphite and 32 isolates from eight orchards treated with phosphite for 15 – 37
years. Four isolates had increased tolerance to phosphite and two were
intermediately tolerant. These six isolates were from phosphite managed orchards
and five were isolated from unhealthy trees.
To test if in vitro tolerance would be expressed in planta, three tolerant and three
sensitive isolates were tested for their ability to colonise phosphite treated lupin
(Lupinus angustifolius) roots. The tolerant isolates were more extensive colonisers
of lupins treated with 5 and 10 g/L phosphite. In the presence of phosphite the
tolerant isolates were able to produce more sporangia and release viable
swimming zoospores more often.
Internationally important Phytophthora species from culture collections in New
Zealand and the United States were assessed for sensitivity to phosphite and a
subset of 32 isolates from the Berkeley collection was tested for mefenoxam
sensitivity. The causal agent of kauri (Agathis australis) dieback, P. agathidicida,
was found to be highly sensitive to phosphite while the possibly New Zealand
native P. kernoviae, was found to be relatively tolerant. Intraspecific variation was
found in lineages of P. ramorum, the causal agent of sudden oak death in the
United States and the United Kingdom, NA2 was more tolerant than NA1. One P.
megasperma isolate was more tolerant to mefenoxam compared to the other
isolates tested. Of nine P. cinnamomi isolates from avocado orchards in southern
California, one was relatively more tolerant to phosphite.
This study has shown phosphite tolerant isolates of P. cinnamomi have developed
in phosphite managed avocado orchards in New Zealand and interspecific
variation in sensitivity to fungicides is present across Phytophthora species in
New Zealand and the United states.
... Resistance gene analogues have been used to study the genetics of resistance in plant species using R gene specific or degenerate primers (Leister et al. 1996;Chen et al. 1998). Azhar et al. (2010) identified 24 RGAs of the NBS class in cotton and utilized these RGAs as markers in interspecific hybrid selection for analysing G. arboreum genome contribution. ...
Cotton leaf curl virus (CLCuV) disease is one of the major limiting factors in cotton production, particularly in widely cultivated Gossypium hirsutum varieties that are susceptible to attack by this virus. Several approaches have been employed to explore putative resistance genes in another cotton species, G. arboreum. However, the exact mechanisms conferring disease resistance in cotton are still unknown. In the current study, we used various approaches to identify possible resistance genes against CLCuV infection. We report the identification and isolation of a set of genes involved in the resistance response to viral infestation. PCR products containing genomic DNA gave multiple amplifications with a single primer in most reactions, and 38 fragments were cloned from G. arboreum and G. hirsutum. The sequences of cloned fragments belonged to various pathway genes and uncharacterized proteins. However, five amplified fragments (RM1, RM6, RM8, RM12 and RM31) showed similarity with R genes. Maximum homology (94 %) was observed with G. raimondii toll/interleukin receptor-like protein. BLAST search showed the homology of all resistance gene analogues (RGAs) with more than one chromosome, and multiple hits were observed on each chromosome for each RGA. Expression analysis through RT–PCR identified variable expression levels of the different RGAs in all tested genotypes. The expression level of RGAs differed between symptomatic and asymptomatic plants, with the exception of RGA 395, whose expression level was the same in both diseased and healthy plants. Knowledge of the interaction of these genes with various cotton pathogens could be utilized to improve the resistance of susceptible G. hirsutum and other plant species.
... Resistance gene analogues have been used to study the genetics of resistance in plant species using R gene specific or degenerate primers (Leister et al. 1996;Chen et al. 1998). Azhar et al. (2010) identified 24 RGAs of the NBS class in cotton and utilized these RGAs as markers in interspecific hybrid selection for analysing G. arboreum genome contribution. ...
Cotton leaf curl virus (CLCuV) disease is one of the major limiting factors in cotton production,
particularly in widely cultivated Gossypium hirsutum varieties that are susceptible to attack by
this virus. Several approaches have been employed to explore putative resistance genes in
another cotton species, G. arboreum. However, the exact mechanisms conferring disease
resistance in cotton are still unknown. In the current study we used various approaches to
identify possible resistance genes against CLCuV infection. We report the identification and
isolation of a set of genes involved in the resistance response to viral infestation. PCR products
containing genomic DNA gave multiple amplifications with a single primer in most reactions, and
38 fragments were cloned from G. arboreum and G. hirsutum. The sequences of cloned
fragments belonged to various pathway genes and uncharacterized proteins. However, five
amplified fragments (RM1, RM6, RM8, RM12 and RM31) showed similarity with R genes.
Maximum homology (94%) was observed with G. raimondii toll/interleukin receptor like protein.
BLAST search showed the homology of all resistance gene analogues (RGAs) with more than one
chromosome, and multiple hits were observed on each chromosome for each RGA. Expression
analysis through RT-PCR identified variable expression levels of the different RGAs in all tested
genotypes. The expression level of RGAs differed between symptomatic and asymptomatic
plants, with the exception of RGA 395, whose expression level was the same in both diseased
and healthy plants. Knowledge of the interaction of these genes with various cotton pathogens
could be utilized to improve the resistance of susceptible G. hirsutum and other plant species.
Keywords: CottonGen, ESTs, Expression, Symptomatic, Asymptomatic, motifs
... Plant resistance genes characterized by the conserved motifs/ domains, have led to designing and synthesis of primers for resistance gene analogue (RGA) markers. The RGA markers were initially designed to map or isolate R genes in potato (Leister et al., 1996) and afterwards successfully used in other crops (Chen et al., 1998;Yan et al., 2003;Mutlu et al., 2006;Que et al., 2009;Hameed et al., 2015). Apart from other DNA markers, the resistance gene analoguemarkers require high resolution electrophoresis and visualization.Sequencing of PCR (polymerase chain reaction) amplified products based on conserved domains of R gene, is employed to isolate molecular markers supposed to be responsible for disease resistance, which is a fast and much less expensive approach. ...
Sugarcane is a key cash crop of India and many other countries, which is grown in subtropical and tropical regions of the world. Around 70% of world’s total sugar comes from sugarcane and its demand is constantly increasing with burgeoning world population. The production and productivity of sugarcane suffers due to various abiotic and biotic stresses. Among
biotic stresses, diseases like red rot, smut, wilt and phytoplasma are major problems. Plant disease resistance (R) genes play
important role in disease control. They provide resistance against various diseases across diverse plant species and they
have certain conserved domains across the plant genera which include nucleotide-binding sites (NBS), leucine-rich repeat
(LRR), serine/threonine protein kinases (PKs) and trans-membrane domains arranged in various combinations. These conserved
sequences have led to the development of novel PCR-based approach for isolation of resistance gene analogues (RGAs)
and, identification of candidate genes for disease resistance from various crop plants. Apart from isolating the resistance
genes, the resistance gene analogue polymorphism (RGAP) has been found as an effective tool to identify molecular markers
linked to disease resistance genes as well as gene diversity analysis in sugarcane. These RGAP markers are equally useful in
development of resistant cultivars and to understand the mechanism of disease resistance and management. Such potential
RGAP markers can be used to develop biotic stress related resistant genotypes via crosses between wild types and cultivated
elite cultivars.
Key words : Disease resistance, NBS-LRR, resistance gene candidates, RGA, red rot, Saccharum.
... The fixed primer, QHB14G14b (aat ctc aag gac aaa agg) is from a sunflower (Helianthus annuus L.) expressed sequence tag (EST) with no homology to any known genes (Hu et al. 2005). The fixed primer F4RGC (ggi ggi gti ggi aai ac, a degenerate primer where ''i'' pairs with any nucleotide) (Leister et al. 1996;Gedil et al. 2001;Kwon et al. 2013) is a resistance gene analog. The random primers, Sa12 (ttc tag gta atc caa caa ca) and Ga5 (gga acc aaa cac atg aag a), were labeled with infrared dyes, IRD700 and IRD800 (Eurofins MWG Operon, Huntsville, AL), respectively. ...
We studied the genetic relationships of Lactuca georgica samples originating in Armenia and the Russian Federation with samples representing four other predominantly self-pollinating wild Lactuca species (L. serriola, L. aculeata, L. saligna, and L. virosa) originating in various countries, as well as with samples representing cultivated lettuce, L. sativa by using 48 TRAP markers. We also visualized their genetic diversity and structure. The present study is likely the first molecular phylogenetic evaluation of a detailed screening of L. georgica germplasm. Data analysis of the three major wild species in this study, L. georgica (134 samples), L. virosa (57 samples), and L. serriola (40 samples) showed that allele frequencies of all 47 polymorphic loci varied significantly among the species. A total of 11, 9, and 10 alleles were unique to L. georgica, L. serriola, and L. virosa, respectively; 71% of TRAP marker diversity was between species. The Neighbor-Joining tree clearly clustered the whole set of 238 samples according to their taxonomic determination. It also reflects the gene diversity as well as the genetic distance values among samples representing the between and within variance of the various species. The L. georgica samples clustered most distantly from the L. sativa samples. The interspecies comparisons between samples belonging to L. georgica with those belonging to L. sativa displayed a high distance, lower only from the interspecies comparisons between samples belonging to L. virosa (in the tertiary gene pool of cultivated lettuce) with those belonging to L. sativa. Thus, additional molecular data with more hybridization experiments are necessary to reconsider if L. georgica is indeed a constituent of the primary gene pool of cultivated lettuce. The L. georgica samples were divided into two sub-clusters, with samples collected in southeast and central Armenia grouping together while all those collected in the north and Dagestan grouped together.
... Most R gene isolates so far contain structural features nucleotide binding domain, leucine-rich repeats and serine/ threonine protein kinase in different combinations (Staskawicz et al. 1995). This similarity among R genes has made it possible to isolate resistant gene candidates from any crop species using a candidate gene (CG) approach (Michelmore 1996;Kanazin et al. 1996;Leister et al. 1996;Shen et al. 1998;Reddy et al. 2010). The use of DNA markers will help to identify nematode resistant chilli germplasm for breeding purposes. ...
Chilli or hot pepper (Capsicum annuum L.), a major commercial vegetable crop of India is challenged with a number of pests and diseases and considerable economic damage, caused by root knot-nematode (RKN). Use of cultivars resistant to RKN would obviate the need for the use of pesticides. More than 40 resistant genes have been reported so far in various crops based on a candidate gene approach to address various biotic stresses. In the present study, isolation of these resistance gene candidates (RGCs) has been performed using a PCR-based approach involving degenerate primers designed based on the conserved nucleotide binding site (NBS) domains. Of the 41 clones sequenced, 9 NBS sequences with complete open reading frames (ORFs) had high similarity with the known resistance (R) genes, and are denoted as CaRGCs (Capsicum annuum resistant gene candidates). The presence of internal conserved motifs provided evidence that sequences isolated from chilli may belong to the NBS-LRR family. A cluster analysis based on the deduced amino acid sequence was carried out on chilli NBS-sequences, together with several analogous domains of known R genes, allowing chilli sequences to be classified into three major groups. The ratio of non-synonymous to synonymous nucleotide substitution (dN/dS) in the NBS domains of chilli RGCs ranged from 0.064 to 0.38 for the different classes, which suggests purifying selection. We report that the partial coding sequence CaRGC6, which shows similarity to a nematode resistant protein, could have a potential advantage in the development of a transgenic chilli provided the full length gene is isolated and characterized.
... Increasingly detailed genetic maps have been developed over the last 30 years (Bonierbale et al. 1988;Gebhardt et al. 1989;Gebhardt et al. 1991;Tanksley et al. 1992;Van Os et al. 2006). Functional maps for genetic factors controlling traits of interest have also been developed (Leister et al. 1996;Grube et al. 2000;Chen et al. 2001;Gebhardt and Valkonen 2001;Bradshaw et al. 2008). There have also been a substantial number of studies aimed at locating genes or quantitative trait loci (QTL) for traits of interest, although they have mainly been performed on diploid populations. ...
A conventional potato breeding strategy uses targeted outcrossing, followed by phenotypic recurrent selection over a series of generations to identify improved cultivars. This paper reviews recent research in Australia aimed at improving the efficiency of such breeding. To develop marker-assisted selection (MAS) for traits of interest, our initial targets were qualitative disease resistances for potato cyst nematode (Globodera rostochiensis Ro1), Potato virus Y and Potato virus X. We undertook a cost analysis comparison between MAS and conventional screening, confirming that MAS would be cost-effective within a breeding programme. Then, as the majority of target traits are quantitative in nature, we also looked at methods to address these traits, including progeny testing and a quantitative genetic analysis technique to develop estimated breeding values (EBVs). We found the markers were useful for detecting the disease resistance characters, while the EBVs improved the analysis of the complex traits. Using a combination of MAS, EBVs and conventional screening methods, we then designed a breeding scheme for rapid selection of cultivars with multiple desirable traits, reducing the breeding cycle from over 10 to 4 years. We then explored the factors that will affect the application of genomic selection in potato and investigated strategies to incorporate genomic selection in potato breeding, as we found that it would accelerate genetic gain as the breeding cycle can be reduced to 1 year. Improvements in computational power are also flowing on to research capabilities such as sequencing, high-throughput phenotyping and data analysis, which will accelerate germplasm improvement and breeding. High-throughput phenotyping facilities are being developed that include automated glasshouse systems equipped with imaging sensors and in-field high-throughput phenotyping systems with sensors mounted on ground- or aerial-based vehicles. Using these technological improvements in phenotypic and genotypic analysis will reduce the breeding cycle in a cost-effective manner and means that we can now breed differently.
... In the mid-1990s, PCR-based methods emerged for the amplification of disease resistance gene sequences from plant DNA, with initial reports from potato (Leister et al. 1996), soybean (Yu et al. 1996;Kanazin et al. 1996), A. thaliana (Aarts et al. 1998), and maize (Collins et al. 1998). Since the NB-ARC domain has motifs such as the P-loop, Kinase-2, and GLPL that are highly conserved among NB-LRR genes ( Fig. 6.1), degenerate PCR primers designed to target those motifs will amplify a set of related sequences, called Resistance Gene Analogs (RGAs), fragments derived from putative genes with predicted R gene function. ...
Plants possess sophisticated surveillance and response systems against potential pathogens. In most cases, the genes underlying plant disease resistance encode nucleotide-binding leucine-rich repeat (NB-LRR) proteins. While the single gene nature of NB-LRR genes makes them widely accessible for plant improvement, the potential for rapid pathogen adaptation and, thus, reduced resistance durability is high. Few disease resistance genes of known function have been cloned in the Rosaceae but several have been mapped, with associated markers available for marker-aided selection. In strawberry, resistance to red stele root rot (Phytophthora fragariae var. fragariae), anthracnose (Colletotrichum acutatum), and angular leaf spot (Xanthomonas fragariae) have all been the targets of genetic mapping. In Rubus, gene H conditions pubescence in raspberry is associated with resistance to gray mold (Botrytis cinerea), spur blight (Didymella applanata), cane blight (Leptosphaeria coniothyrium), and cane spot (Elsinoë veneta). It is unclear whether pubescence acts as a preformed physical barrier to infection or if gene H is physically linked to NB-LRR genes conditioning the various resistances. Resistance to Raspberry bushy dwarf virus has been genetically mapped and markers associated with resistance to the aphids Amphoromphora idaei and Amphorophora agathonica, vectors of important raspberry viruses, have been identified. Candidate gene approaches including PCR-based methods for generating resistance gene fragments hold some potential for development of markers useful in strawberry and raspberry breeding. Finally, the availability of whole genome sequences from Fragaria and Rubus species enables in silico discovery of NB-LRR genes and visualization of evolutionary relationships and physical genome distribution, a focus of research in our research laboratory.
... Fragment sizes ranged from 300 bp to 1.2 kb. Those targeted candidate genes are involved in herbaceous plants in: Ø gene-for-gene disease resistance (RGA resistance gene analogs or R genes from the LRR and protein kinase families) (LEISTER et al. 1996), similarly to the N gene product which belongs to the NBS-LRR family and confers resistance to Tobacco mosaic virus Ø defence response (transcription factors, protein kinases, PR proteins) and genes coding for proteins involved in the salicylic acid pathway and hypersensitive reaction Ø viral RNA silencing similar to post-transcriptional gene silencing, another type of natural defence mechanisms against viruses in plants ( MATZKE et al. 2001). Some other candidates were targeted, they are coding for proteins interacting with the virus and tightly implicated in the virus replication and host invasion (i.e. ...
Plum pox virus (PPV), the causing agent of the sharka disease, belongs to the genus Potyvirus that contains the largest number of virus species infecting plants. The virus genome has been extensively characterised and sequenced. However, few data are available on its interactions with the host plant, Prunus. In this study, we are focusing on the cloning and characterisation of any candidate genes involved in the expression of the resistance/susceptibility trait and any polymorphic genes putatively involved in the trait variation. In order to clone candidate genes, two main approaches are currently developed: the homology cloning of genes presumed to affect the resistance/susceptibility trait and the differential screening of cDNA pools corresponding to infected and non-infected plant material. The second approach is based on the transcript imaging of the host plant response to PPV infection. Previously, it has been shown that infection by a potyvirus is associated with specific changes in host gene expression, mainly down-regulation, while the expression of some genes remained unchanged. Thereby, in the differential display approach combined to further characterisation of candidate gene expression, we aim to monitor host gene expression in response to the virus and to describe a highly regulated interaction between the Prunus host plant and the infecting Plum pox virus.
The presence of various micro-organisms causing diseases poses a significant threat to crop cultivation, leading to reduced economic yields. Plant ailments can lead to yield losses of around 40% or even more, implying that only about 60% of the crop's economic potential is realized, with the rest compromised by diseases. Researchers have studied the molecular cloning of R genes, which confer resistance to a wide array of pathogens, and have discovered common characteristics among the proteins encoded by these genes. Breeding crops for resistance is seen as the most efficient and practical method of control, as it avoids additional costs and is environmentally friendly. In the pursuit of disease-resistant genes, molecular techniques have proven to be highly effective, requiring less time and effort for identification, isolation, cloning, and transfer of these genes between different species. Molecular geneticists are now emphasizing the mapping and tagging of disease resistance genes, intending to share this information within the global scientific community.
Microsatellites or simple sequence repeats (SSRs) markers are a helpful strategy among the various molecular marker techniques for strengthening molecular breeding programs. These are useful markers for genotyping plant populations with tandem repeats of 2-6 base pair length DNA patterns. The expansion of various molecular markers and advances in sequencing technologies has aided crop improvement. Several articles for research scholars with progressive knowledge of molecular genetics have been published in the last three decades to probe the information regarding molecular markers. This article reviews novel advances regarding molecular markers and their implementations in plant breeding for researchers with no expertise in using molecular markers in plant breeding. A superior comprehension of molecular markers, and a better consciousness of the spectrum of crops that can be grown, has resulted from progress in molecular plant breeding, genetics, genomic selection, and genome rectification. Further-generation sequence technology must enable the production of novel genetic markers for multifaceted and amorphous groups through genotyping-by-sequencing and union mapping. The review also discusses almost all the microsatellite markers and their advantages and disadvantages.
At different stages of host–pathogen interactions, natural conditions and modern agricultural settings foster the evolution of new and virulent pathogenic races by selection pressure on R genes in crucifer genotypes. Polyploidy in Albugo candida contributes to race diversification and evolution by selection pressure imposed from cultivated Brassica species. Molecular studies have revealed high degree of genetic diversity within Albugo pathogenic to Brassica species with wide host range and six new specialized species along with virulent races. ITS and COX2, molecular approaches of phylogenetic analysis and morphological variations of oospores have divided A. candida into three groups (1, 2, 3) infecting different cruciferous with different races. Several races/pathotypes of A. candida have been identified at global level infecting different crucifers. The virulence of A. candida races varies from very narrow to wide infecting different Brassica species and genotypes. Phylogenetic analysis and nucleotide sequences have distinguished races of A. candida infecting different crucifers in different countries of the world. Albugo candida isolates from different crucifers have been characterized based on virulence genes specificity to their hosts in Australia. A single dominant gene AvrAC1 controls avirulence in race 2 of A. candida infecting B. rapa cv. Torch. The genetic variation among geographically distinct isolates of A. brassicae has been assessed using RAPD-PCR markers. The genetic structure of A. brassicicola population indicates the occurrence of sexual recombination’s with a cryptic sexual stage. Mutation of Amr1 gene in A. brassicicola causes increased virulence. Three pathotypes of A. brassicae viz., RM1, RM2, and V3 have been identified using eight commonly cultivated Brassica species. The isolates of A. brassicae collected from different geographical areas are different in conidial morphology, cultural characteristics, and symptom production on different hosts. A brassicae isolates from different locations show high degree of genetic similarity. There is differential protein expression by the isolates of A. brassicae. The isolates of A. brassicae show variability in biochemical contents, sensitivity to different fungicides and plant extracts. The variation in relative levels of virulence in the form of disease incidence, severity, and defoliation within and between Alternaria spp. causing leaf spot disease on rapeseed-mustard has been recorded. Several pathotypes of Alternaria species have been identified with different designations using differential determinant attributes. The function of effectors/genes, ChELP1, and ChELP2 homologs of Lysm proteins has been characterized in C. higginsianum. In India, powdery mildew pathogen produces both asexual and sexual stages on B. juncea, so there is every possibility for Erysiphe cruciferarum to express pathogenic variability. The isolates of Hyaloperonospora parasitica collected from different countries, locations, and host species have been characterized in the form of pathotypes showing host specificity and differential virulence. The isolates of H. parasitica from different host species and genotypes have been molecularly identified as pathotypes with their virulence potential using DNA (RAPD) finger printing. The isolates of H. parasitica collected and characterized on different crucifers as race and pathotypes cannot be compared in the absence of use of standardized host differentials (isogenic lines) and uniform designation/nomenclature system at international level. Hybridization of Hyaloperonospora isolates has also been observed. The evolution of virulent races in Leptosphaeria has been observed in the areas where crops with major gene resistance coupled with genetic uniformity have been sown. The avirulence genes of L. maculans races have been identified from Europe. Leptosphaeria maculans field populations have high level of gene diversity and genotypic diversity in France. An avirulent mutated AvrLm gene of L. maculans has been identified to detect new R genes for breeding blackleg resistant cultivars of Brassica. The genome-wide DNA variants and SNP haplotypes of L. maculans pathotypes have been identified. Pathotypes of Plasmodiophora brassicae has been identified from various countries using different differential host sets and designating pathotypes in their own way without following any sound uniform system of nomenclature. The genomes of P. brassicae pathotypes Pb2, Pb5, and Pb8 have been sequenced to gain insight into genome variations and its correlation with host specificity. Proteomics of P. brassicae has revealed potential effectors/genes of pathotypes. The isolates of P. brassicae from different countries have been phylogenetically analyzed for similarities and differences of pathotypes. Polymorphism in P. brassicae in case of Korean isolates collected from field has been recorded. The Cr811 gene in P. brassicae pathotypes P5 plays an important role as molecular markers for identification of race P5 and other new virulences. The different geographical isolates of P. brassicae have been identified by rDNA sequence polymorphism. Pathogenic and genetic variations in P. brassicae pathotypes infecting a weed C. flexuosa and crucifers have been analyzed using RAPDs in Japan. The virulence of P. brassicae changes during interaction with CR genes in the host cultivars. The changes in P. brassicae pathotypes structure under field conditions have been analyzed through whole genome DNA similarity sequences. The application of RAPD seq has revealed distinct P. brassicae populations under Canadian conditions. ITS sequencing and phylogenetic analysis of P. capsellae isolates from Brassica hosts clearly differentiated them in to virulence factors/races. High level of genetic diversity and recombination’s in Sclerotinia populations in tropical countries has been identified with the groups of isolates as MCGs and MLHs infecting crucifers. The aggressiveness of Sclerotinia isolates on Brassica has been measured using AUDPC as one of the parameters. The sequence variation of ITS region of S. sclerotiorum isolates revealed higher heterogenecity and genetic variability showing presence of clonal and sexual progenies of the pathogen in B. juncea growing areas of India. The genetic diversity is higher in Sclerotinia populations from Canola and Sunflower in the NCUS. Distribution of microsatellite haplotypes regions is often on multiple crops. The genetic diversity and populations structure of S. sclerotiorum has been analyzed with DNA (RAPD) sequences from different countries of the world. The virulence factors of Sclerotinia has been identified on the basis of pathogenicity as virulent pathotypes (aggressiveness), genomic factors (dsRNA) controlled Hypo virulence, MCGs, cloned variables, haplotypes, genetic diversity of isolates, and population biology studies with markers such as MCGs, DNA finger printing or micro satellites. The TuMV isolates collected from different hosts could not be characterized on Brassica species for virulence. The molecular characterization of isolates from different species of Verticillium indicated that isolates infecting crucifers are long spored (amphihaploids or allodiploids) because of Verticillium interspecific hybridization events. Black rot of crucifers’ bacterium has been characterized into pathovars of X. campestris on different species of crucifers. The X. campestris isolates from oilseed rape are more genetically diverse with specialization to their nonhost than their Brassica hosts. The virulent effectors/genes of pathogens have different evolutionary mechanisms to counteract with the defense mechanisms of the host. The identification, classification, and utilization of crucifer’s pathogen pathotypes from different countries is most challenging task since scientists have not followed standardized uniform system and procedures in selecting host differentials, designation and nomenclature of pathotypes, pure isolates (single spore), and scoring of infection phenotypes. The expression of high genetic diversity by crucifers’ pathogens under natural as well as modern agriculture settings is another challenge of variation in pathogenic populations.KeywordsPathogenomicsPathogenic variabilityAlbugo PathogenomicsPolyploidy to Albugo race diversificationPhylogenetic relationshipVirulence spectrum of virulence genesInheritance of avirulenceAlternaria pathogenomicsGenetic variationMutation of Amr1 geneEvolution of virulenceIdentification of pathological variationsColletotrichum pathogenomicsErysiphe pathogenomicsIdentification of pathotypesHyaloperonospora pathogenomicsIdentification of virulence effectors/genesHeterothallism and homothallismLeptosphaeria pathogenomicsEvolution of virulenceAvirulence allelesGenetic variabilityCharacterization of avirulent mutated AvrLm geneIdentification of genome-wide DNA variantsSNP haplotypePlasmodiophora pathogenomicsDifferentials for identification of pathotypesGenome comparison of pathotypesProteomics of pathotypesPhylogenetic analysis of pathotypesMolecular marker for identificationIdentification of geographical isolatesPathogenic and genetic variationWhole genome DNA of pathotypePseudocercosporella pathogenomicsIdentification of virulence factors/racesSclerotinia pathogenomicsGenetic diversityStructureRandom amplified polymorphic DNA markersTurnip mosaic virus pathogenomicsPathological and biological characteristicsVerticillium pathogenomicsXanthomonas pathogenomicsIdentification of pathotypesEvolution of virulent effectorsChallenges of pathotype identificationGenetic variation
Crucifer’s crops are very important crop commodity with very significant contribution in the world’s need of human and animals yielding edible and industrial oil along with vegetables and forage crops of economic, trade, and food security. These groups of crops are challenged by numerous pathogens threatening their production at global level. Some of them have been exploited to reveal genomics of host–pathogen interaction to comprehend the molecular mechanisms of infection and pathogenesis for better management of diseases they cause and influence yield and quality losses. The genome of six Brassica crops has been sequenced after the sequencing of Arabidopsis thaliana genome. The genome size of A. thaliana is smallest with 125 Mb and B. napus genome is largest with 925 Mb. To get deeper insight into the molecular and biological functions of host–pathogen interaction, genome of major pathogens of crucifers has been sequenced and analyzed. Among pathogens, P. brassicae genome is compact and smallest with 24.2–25.5 Mb and G. orontii genome causing powdery mildew is largest with 160 Mb in size. The genome analysis of pathogens has facilitated phylogenetic, host specificity, pathogenicity factors/genes, and molecular events during crucifer’s host–pathogen interactions. The perception and understanding of molecular and genetical mechanisms of host–pathogen recognition and events of pathogenesis are regulated by genomics modulation of interacting host and pathogen. Pathogenomics have revealed host specificity, change of host range, and evolution of pathogenic variability. The R genes regulate functions, and molecular mechanisms of host–pathogen interactions. The timing of the induction of genes in R, and S varieties is crucial for mounting effective host defense to the pathogens. Transcriptomic analysis has revealed genes strongly associated with pathogenesis. The genes involved in cell response signaling, cell wall degradation, protein degradation, enzymes production, host transcriptional, and hormonal regulation are differentially expressed. The virulence mechanisms are transition period from biotrophy to necrotrophy to facilitate the acquisition of host nutrients by the pathogen. Differential expression of up and down regulated genes, and functional groups has been identified during host–pathogen interaction. The whole genome analysis of host and pathogen offers potential unbiased insight into the molecular mechanisms of host-pathosystem in crucifers.For rapid, accuracy, less laborious, and less expensive way of detection and identification of crucifer’s pathogens, pathotypes, and effectors genes, molecular protocols have been used at field, and controlled laboratory conditions. The distinct lineages that had diverged from each other have also been detected. Molecular markers (RAPD, ESTs) have been used for detection and identification of genetical, and pathological variation and clades of pathogens. The molecular approaches have been allowed detection, identification, and quantification of pathogens host cell entry, area covered by pathogens in host tissues, seed, and soil. The RT-PCR protocols have been developed to detect, and discriminate AGs of R. solani from field, soil, and viruses along with their strains from Brassica species. High degree of genetic diversity has been recorded in major pathogens of crucifers in the form of pathotypes/races/strains differing in virulence, and host range on Brassica species and genotypes. The evolution of new virulence’s of pathogens is more common in areas where crops with major gene resistance coupled with genetic uniformity have been sown. The genomes of P. brassicae pathotypes Pb2, Pb5, and Pb8 have been sequenced to gain insight into genome variations and its correlations with host specificity. Phylogenetics of pathotypes has been assessed. The changes in pathotypes structure under field conditions have been analyzed through whole genome DNA similarity sequences. The infection of pathogens in Brassica species activates host metabolism to regulate carbohydrates, respiration, lipid profile, enzymes, toxins, H2O2, OH, phenols, hormones, nucleic acid, proteins, electrolytes, GSL, ROS, and other metabolites which affects crucifer’s physiology, biochemistry, and molecular events leading to pathogenesis. Several genes are differentially expressed during host–pathogen interaction for virulence at different stages of host infection and disease development. The genomics of crucifers host-pathosystem has been studies with simple, reproducible, and standardized methodology to elaborate genome sequencing of host as well as pathogen, events of host and pathogen interaction, signaling pathways, expression of different genes, analysis of transcriptome, biochemical changes, pathogenic variability, molecular markers, transition period from biotrophy to necrotrophy, detection, and identification of pathogens, pathotypes, and genes during the process of infection and pathogenesis of crucifer cops.KeywordsCrucifer’sPathogensGenomicsGenome sequencingEvents of host–pathogen interactionOmics technologiesSignaling pathwaysQRT-PCRMolecular functionsChoice of reference genesPathogens genome assemblyHost–pathogen recognitionPathogenomicsDifferential proteins/genes expressionHost-transcript regulationTransition from biotrophy to necrotrophyHormonal regulationGene expressionSpliceosome genesEvolutionary genomicsMolecular detection and identification of pathogensPathotypes and genesMolecular markersGenomic prediction toolsCorrect diagnosisSelection pressureRace virulenceHost differentialsProteomicsAggressivenessHypovirulenceMetabolomicsToxinsHost-biochemistryAltered metabolismProtocolsMethodologyTechniques
The genus Solanum includes important crop species and numerous wild species. The wild Solanum species are a genetic diversity treasure trove useful for the improvement of crops, with disease resistance being of critical importance. In this chapter, I review the status of research and breeding efforts for improved nematode and virus resistance in Solanum crops, especially potato and tomato. In total, 33 disease resistance genes are described; virtually all originate from wild relatives of potato and tomato. This observation underscores the utility of wild Solanum species and the need to prioritize their conservation through in situ and ex situ approaches. Trends in research well positioned to impact trait discovery in wild Solanum species and introgression into crop species are outlined. Included are the potential to mine genebank collections for novel disease resistance alleles using target DNA sequencing approaches, visualization of deep evolutionary and allele diversification patterns across the Solanaceae, and streamlined gene mapping and cloning methodologies. The potential impacts of Marker Assisted Breeding, genetic transformation, gene-editing, and conversion of cultivated potato to a diploid species are explored. A growing world population and changing global climate that requires crop plants to tolerate increasingly chaotic production environments present the need for urgent investment in the genetic improvement of crops, with crop wild relatives being critical donors of useful traits.
The characterization and selection of molecular markers are important for genetic pre-breeding programs since they make it possible to choose the most appropriate markers to be used in future research. Therefore, enabling the generation of subsidies for genetic-molecular studies in algabora (Prosopis juliflora (Sw.) DC). The amplification profile was characterized. It was generated from 17 pairs of RGA primers (Resistance Gene Analogs) in 20 samples of genomic DNA of P. juliflora extracted from specimens collected in the city of Itapetinga, Bahia. The amplifications were performed according to previously published laboratory routines and the amplification profiles analyzed from the photodocumentation of the electrophoresis results in 2% agarose gels. Based on the amplification profiles the primer pairs were classified as: Suitable: amplifications in the whole samples and with easy visualization; Reasonable: amplification in parts of the samples and/or difficult to visualize or Inadequate: absence of visible amplification products. Descriptive analyzes associated with the number of generated markers, percentage of polymorphism, expected heterozygosity (He) and the content of polymorphic information (PIC) were also performed. In a nutshell, 12 out of the 17 pairs of RGA primers generated amplification products with easy visualization and only two of these 12 pairs of primers were monomorphic. The percentage of polymorphism varied from 60% to 100%, He and PIC presented an average of 0.21 (ranging from 0 to 0.38) and 0.17 (ranging from 0 to 0.29), respectively. The results confirm that the RGA primers present adequate characteristics for genetic studies in P. juliflora, making it possible to prioritize 12 pairs of primers, which are subject to genetic improvement studies.
The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genome of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDA's varieties differ from widely available medical, recreational, or industrial Cannabis. Furthermore, our results suggest that NIDA's varieties lack diversity in the single copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome. Therefore, results based on NIDA's varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation.
The sections in this article are
Introduction
Features of Cloned Resistance Genes
Control of Resistance Gene Specificity
Do Resistance Proteins Interact Directly with Avirulence Determinants?
Organisation of Resistance Gene Loci
Evolution of Resistance Genes by Divergent Selection
Evolution of Resistance Genes by Recombination
Concluding Remarks
The sections in this article are
Introduction
History of Late Blight
Economic and Social Impact of P hytophthora Plant Pathogens
P hytophthora Infestans and its Taxonomic Position
The Disease Cycle of P hytophthora Infestans
The Plant Response
Future Perspectives
Acknowledgements
Plant-parasitic nematodes are microscopic roundworms that live in many habitats. They cause substantial problems to major crops throughout the world, including vegetables, fruits, and grain crops. These may become a major threat to the agricultural production system worldwide if management fails. This chapter reviews the economic importance and diagnostic methods of plant-parasitic nematodes, including a comprehensive account of existing strategies used for their management ranging from conventional to modern techniques. Some important genera of plant-parasitic nematodes such as Meloidogyne spp., Heterodera spp., and Pratylenchus spp. have been ranked uppermost in the list of the most economically and scientifically significant species of nematodes due to their complicated relationship with the host plants, wide host range, and the level of damage due to infection in crops. Further, obstacles encountered in parasitic nematode diagnosis by classical morphology-based methods have been resolved by the adoption of novel molecular techniques, which are rapid, precise, and cost-effective. As far as the existing cultural management techniques are concerned, crop rotation with non-host crops can suppress a wide range of nematode species effectively, followed by the use of organic soil amendments. Nematicide application is effective when speedy control of nematodes is required; however, the use is reappraised due to environmental concerns. Biological control of nematodes by fungi and bacteria is highly favored due to its environmentally friendly nature. In addition, bio-pesticides are becoming a promising option for the management of plant-parasitic nematodes. Biotechnology-, molecular biology-, and nanotechnology-based approaches have added a new dimension to nematode disease diagnosis and management. Identification of genes that reduce nematode’s ability to reproduce has allowed the breeding of nematode-resistant plants. Marker-assisted selection, genetic engineering, and RNA interference to confer resistance in crop plants, nematode suppression using host plant proteinase inhibitors, and genome-editing technologies have helped tremendously in developing management strategies for plant-parasitic nematodes. In conclusion, a sustainable management of plant-parasitic nematodes is feasible when two or more compatible tactics are applied concurrently while appraising environmental protection.
Black pepper is an important commodity crop in Malaysia that generates millions of annual revenue for the country. However, black pepper yield is affected by slow decline disease caused by a soil-borne fungus Fusarium solani. RNA sequencing transcriptomics approach has been employed in this study to explore the differential gene expression in susceptible Piper nigrum L. and resistant Piper colubrinum Link. Gene expression comparative analysis of the two pepper species has yielded 2,361 differentially expressed genes (DEGs). Among them, higher expression of 1,426 DEGs was detected in resistant plant. These DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). We selected five groups of defence-related DEGs for downstream qRT-PCR analysis. Cf-9, the gene responsible for recognizing fungal avirulence protein activity was found inexpressible in susceptible plant. However, this gene exhibited promising expression in resistant plant. Inactivation of Cf-9 could be the factor that causes susceptible plant fail in recognition of F. solani and subsequently delay activation of adaptive response to fungal invasion. This vital study advance the understanding of pepper plant defence in response to F. solani and aid in identifying potential solution to manage slow decline disease in black pepper cultivation.
Living beings are dependent on green energy for their survival and crops are playing most potential role in it. Sudden changes, i.e., biotic and abiotic stresses, are always a big factor affecting the productivity of crops, and in this adverse environmental conditions, it is very much imperative to find the alternate strategies for increasing crop productivity and to feed the increasing worldwide populations. We have always the knowledge of obtaining the best cultivar as earlier the farmers used to select the healthy seedlings for agriculture. Gregor Johann Mendel’s work Mendelian inheritance gives us a platform to use the conventional methods with scientific and validated models for better yields of crops. Therefore, initiatives need to be taken to tackle this problem. In recent years advanced biotechnological approaches, tools, and techniques have played important role. With the use of these advanced technologies researchers are able to do whole genome sequencing, annotation, prediction, and validation of gene expression, prediction of possible function as well as their involvement in various metabolic pathways, and identification of the presence of set of nucleic acid sequences (motif) in the promoter region as well as their protein-DNA interaction. Efforts has also been done to develop the genetically modified crop having important characters and to give good yield under adverse conditions.
We studied rDNA restriction fragment length polymorphism between two tomato lines used for F1 hybrid seed production: line A, containing the Tm-1 gene responsible for tobacco mosaic virus tolerance introgressed from the wild species Lycopersicon hirsutum, and line B, a tobacco mosaic virus sensitive line. Hybridization patterns led to distinct rDNA maps with two size classes, 10.4 and 10.7 kb, in line A and a single, 8.9-kb class in line B. Size differences were located in the intergenie sequence (IGS). A highly specific 54-bp TaqI fragment was cloned from the line A IGS and used in dot blot experiments to probe total DNA from line A, line B, and their F1 hybrid. It proved capable of discriminating B from A and the hybrid. This probe could thus serve to screen inbreds in commercial seed lots where line A is used as male. This fragment showed 80-90% sequence homology with the 53-bp subrepeats previously characterized in a region of the tomato IGS close to the 25S rRNA gene. Preliminary comparison of rDNA in line A and several wild related species indicated that the L. hirsutum H2 genotype was the closest to line A. rDNA variations between line A and this wild genotype could be explained by recombination during the introgression process involving numerous backcrosses or by an important intraspecific polymorphism. Our results strongly suggest that Tm-1 and the rDNA were introgressed together into tomato from L. hirsutum through linkage drag.
In the 1940's the root-knot nematode resistance gene (Mi) was introgressed into the cultivated tomato from the wild species, L. peruvianum, and today it provides the only form of genetic resistance against this pathogen. We report here the construction of a high resolution RFLP map around the Mi gene that may aid in the future cloning of this gene via chromosome walking. The map covers the most distal nine map units of chromosome 6 and contains the Mi gene, nine RFLP markers, and one isozyme marker (Aps-1). Based on the analysis of more than 1,000 F2 plants from four crosses, we were able to pinpoint the Mi gene to the interval between two of these markers — GP79 and Aps-1. In crosses containing the Mi gene, this interval is suppressed in recombination and is estimated to be 0.4 cM in length. In contrast, for a cross not containing Mi, the estimated map distance is approximately 5 times greater (ca. 2 cM).
Using RFLP markers around Mi as probes, it was possible to classify nematode resistant tomato varieties into three types based on the amount of linked peruvianum DNA still present. Two of these types (representing the majority of the varieties tested) were found to still contain more than 5 cM of peruvianum chromosome — a result that may explain some of the negative effects (e.g. fruit cracking) associated with nematode resistance. The third type (represented by a single variety) is predicted to carry a very small segment of peruvianum DNA (<2 cM) and may be useful in the identification of additional markers close to Mi and in the orientation of clones during a chromosome walk to clone the gene.
Accessions of the wild tomato species L. peruvianum were screened with a root-knot nematode population (557R) which infects tomato plants carrying the nematode resistance gene Mi. Several accessions were found to carry resistance to 557R. A L. peruvianum backcross population segregating for resistance to 557R was produced. The segregation ratio of resistant to susceptible plants suggested that a single, dominant gene was a major factor in the new resistance. This gene, which we have designated Mi-3, confers resistance against nematode strains that can infect plants carrying Mi. Mi-3, or a closely linked gene, also confers resistance to nematodes at 32°C, a temperature at which Mi is not effective. Bulked-segregant analysis with resistant and susceptible DNA pools was employed to identify RAPD markers linked to this gene. Five-hundred-and-twenty oligonucleotide primers were screened and two markers linked to the new resistance gene were identified. One of the linked markers (NR14) was mapped to chromosome 12 of tomato in an L. esculentum/L. pennellii mapping population. Linkage of NR14 and Mi-3 with RFLP markers known to map on the short arm of chromosome 12 was confirmed by Southern analysis in the population segregating for Mi-3. We have positioned Mi-3 near RFLP marker TG180 which maps to the telomeric region of the short arm of chromosome 12 in tomato.
The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F2 Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F2 interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.
The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich
repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and
subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.
High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker-assisted breeding and evolutionary studies in these two important and well studied crop species.
A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
Plants can recognize pathogens through the action of disease resistance (R) genes, which confer resistance to pathogens expressing
unique corresponding avirulence (avr) genes. The molecular basis of this gene-for-gene specificity is unknown. The Arabidopsis
thaliana RPM1 gene enables dual specificity to pathogens expressing either of two unrelated Pseudomonas syringae avr genes.
Despite this function, RPM1 encodes a protein sharing molecular features with recently described single-specificity R genes.
Surprisingly, RPM1 is lacking from naturally occurring, disease-susceptible Arabidopsis accessions.
Plant breeders have used disease resistance genes (R genes) to control plant disease since the turn of the century. Molecular
cloning of R genes that enable plants to resist a diverse range of pathogens has revealed that the proteins encoded by these
genes have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms
for the expression of resistance to a wide range of unrelated pathogens. Characterization of the molecular signals involved
in pathogen recognition and of the molecular events that specify the expression of resistance may lead to novel strategies
for plant disease control.
Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.
The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.
Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V. The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F1 populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F1 populations analysed, whereas the R1 allele segregated with a 1:1 ratio as expected in five F1 populations. The mode of inheritance of the R10 allele could not be deduced as only very few F1 hybrids bearing R10 were obtained. Linkage analysis in two F1 populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromosome XI.
The products of plant disease resistance genes are postulated to recognize invading pathogens and rapidly trigger host defense responses. Here we describe isolation of the resistance gene N of tobacco that mediates resistance to the viral pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator transposon. A genomic DNA fragment containing the N gene conferred TMV resistance to TMV susceptible tobacco. Sequence analysis of the N gene shows that it encodes a protein of 131.4 kDa with an amino-terminal domain similar to that of the cytoplasmic domain of the Drosophila Toll protein and the interleukin-1 receptor (IL-1R) in mammals, a nucleotide-binding site (NBS), and 14 [corrected] imperfect leucine-rich repeats (LRR). The sequence similarity of N, Toll, and IL-1R suggests that N mediates rapid gene induction and TMV resistance through a Toll-IL-1-like pathway.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
The potato, Solanum
tuberosum, a species of the family Solanaceae, is cultivated in most temperate and subtropical zones of the world. After wheat, rice and corn it occupies the fourth position in terms of world production per year (FAO Yearbook 1988).
During the adaptation of plants to low temperature, changes in gene expression can be induced in a variety of tissues. Low-temperature-regulated gene expression was studied in cold-stored potato (Solanum tuberosum L.) tubers by two-dimensional electrophoresis of in vitro translation products. As a response to cold treatment, the relative amount of mRNA encoding at least 26 polypeptides changed. By differential screening of a cDNA library, 16 clones corresponding to cold-inducible transcripts were isolated. They were classified into four non-cross-hydridizing groups.RNA hybridizations using representative clones from each group revealed different temporal accumulation patterns for the cold-inducible transcripts. mRNAs homologous to the cDNA clones were first detectable after 1 to 3 d of cold treatment, and the highest level of expression was reached after 3 to 7 d. Transcripts corresponding to cDNA clones Cl13 and Cl19 were transiently expressed, whereas the steady-state level remained high for cDNA clones Cl7 and Cl21 during the cold storage period of 4 weeks.The DNA sequences of two cDNA clones, Cl7 and Cl19, have been determined. The polypeptide predicted from the DNA sequence of Cl19 is sequence related to small heat-shock proteins from other plant species. The deduced protein sequence of Cl7 exhibits strong homology to the dehydrin/RAB group of dehydration stress- and abscisic acid-inducible polypeptides and to cold-induced proteins from Arabidopsis and spinach.
A morphologically and agronomically heterogeneous collection of 38 diploid potato lines was analysed for restriction fragment length polymorphisms (RFLPs) with 168 potato probes, including random genomic and cDNA sequences as well as characterized potato genes of known function. The use of four cutter restriction enzymes and a fragment separation range from 250 to 2,000 bases on denaturing polyacrylamide gels allowed the detection of RFLPs of a few nucleotides. With this system, 90% of all probes tested showed useful polymorphism, and 95% of those were polymorphic with two or all three enzymes used. On the average, 80% of the probes were informative in all pairwise comparisons of the 38 lines with a minimum of 49% and a maximum of 95%. The percentage of heterozygosity was determined relative to each other for each line and indicated that direct segregation analysis in F1 populations should be feasible for most combinations. From a backcross involving one pair of the 38 lines, a RFLP linkage map with 141 loci was constructed, covering 690 cMorgan of the Solanum tuberosum genome.
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.
The inheritance and the linkage relationship of resistance to race 3 of Fusarium oxysporum f. sp. lycopersici derived from Lycopersicon pennellii (LA 716) were analyzed in an interspecific backcross to L. esculentum. Progeny from each backcross (BC1) individual were in-oculated with race 3 and their response was measured according to a visual rating system; progeny responses were used to calculate a mean disease rating for each BC1 individual. The frequency distribution of the disease ratings was bimodal, indicating that resistance to race 3 was controlled by one major locus. Linkage analysis of this locus proceeded in two steps. Initially, disease ratings were compared between homozygotes and heterozygotes at each of 17 segregating marker enzyme loci. Highly significant differences were detected for the chromosome segment marked by the Got-2 locus on chromosome 7. This indicated that Got-2 was linked to the race 3 resistance gene, designated I-3; this gene accounted for the observed bimodal distribution of BC1 disease ratings. In the second step, the genotype of each BC1 individual at the I-3 locus was determined using cluster analysis of the disease ratings; a test for independent assortment between I-3 and Got-2 revealed strong linkage, with an estimated map distance of 2.5 centiMorgans. Additional evidence for this linkage was obtained from the analysis of five breeding lines previously selected for resistance to race 3 solely on the basis of inoculations; all five showed co-selection for Got-2. The Got-2 locus is proposed as a selectable marker to expedite the transfer of race 3 resistance to commercial tomato cultivars.
The inheritance and linkage relationships of a gene for resistance to Fusarium oxysporum f. sp. lycopersici race 1 were analyzed. An interspecific hybrid between a resistant Lycopersicon pennellii and a susceptible L. esculentum was backcrossed to L. esculentum. The genotype of each backcross-1 (BC1) plant with respect to its Fusarium response was determined by means of backcross-2 progeny tests. Resistance was controlled by a single dominant gene, I1, which was not allelic to I, the traditional gene for resistance against the same fungal pathogen that was derived from L. pimpinellifolium. Linkage analysis of 154 molecular markers that segregated in the BC1 population placed I1 between the RFLP markers TG20 and TG128 on chromosome 7. The flanking markers were used to verify the assignment of the I1 genotype in the segregating population. The results are discussed with reference to the possibility of cloning Fusarium resistance genes in tomato.
An RFLP linkage map of the potato is presented which comprises 304 loci derived from 230 DNA probes and one morphological marker (tuber skin color). The self-incompatibility locus of potato was mapped to chromosome I, which is homoeologous to tomato chromosome I. By mapping chromosome-specific tomato RFLP markers in potato and, vice versa, potato markers in tomato, the different potato and tomato RFLP maps were aligned to each other and the similarity of the potato and tomato genome was confirmed. The numbers given to the 12 potato chromosomes are now in accordance with the established tomato nomenclature. Comparisons between potato RFLP maps derived from different genetic backgrounds revealed conservation of marker order but differences in chromosome and total map length. In particular, significant reduction of map length was observed in interspecific compared to intraspecific crosses. The distribution of regions with distorted segregation ratios in the genome was analyzed for four potato parents. The most prominent distortion of recombination was found to be caused by the self-incompatibility locus.
Resistance to the root cyst nematode Globodera rostochiensis is an agronomic trait that is at present incorporated into most new potato varieties. Major dominant genes are available that originate from wild and cultivated Solanum species closely related to the cultivated European potato (Solanum tuberosum ssp. tuberosum). One of those genes, H1, from S. Tuberosum ssp. andigena, was mapped to a distal position on potato chromosome V using restriction fragment length polymorphism (RFLP) markers. The H1 locus segregates independently from Gro1, a second dominant gene presumably from S. Spegazzinii that confers resistance to G. Rostochiensis and which has been mapped to chromosome VII. One marker, CP113, was linked without recombination to the H1 locus.
We report the identification and mapping of two quantitative trait loci (QTLs) of Solanum spegazzinii BGRC, accession 8218-15, involved in resistance to the potato cyst-nematode Globodera rostochiensis pathotype Ro1, by means of restriction fragment length polymorphisms (RFLPs). For this purpose we crossed a susceptible diploid S. tuberosum with the resistant S. spegazzinii, and tested the F1 population for resistance to the Ro1 pathotype. Since the F1 segregated for the resistance, the S. spegazzinii parent was concluded to be heterozygous at the nematode resistance loci. For the mapping of the resistance loci we made use of RFLP markers segregating for S. spegazzinii alleles in the F1. One hundred and seven RFLP markers were tested in combination with four different restriction enzymes; 29 of these displayed a heterozygous RFLP pattern within S. spegazzinii and were used for mapping. Analysis of variance (ANOVA) was applied to test the association of the RFLP patterns of these markers with nematode resistance. Two QTLs involved in disease resistance to Globodera rostochiensis pathotype Ro1 were identified and mapped to chromosomes 10 and 11 respectively.
The whitefly-transmitted tomato yellow-leaf curl gemini-virus (TYLCV) is a major pathogen of tomatoes. The wild tomato species Lycopersicon chilense, which is resistant to the virus, was crossed to the cultivated tomato, L. esculentum. The backcross-1 selfed (BC1S1) generation was inoculated and a symptomless plant was selected. This plant was analyzed using 61 molecular markers, which span the tomato genome, to determine which L. chilense chromosome segments were introgressed. A BC2S1 population was cage-inoculated with viroliferous whiteflies (Bemisia tabaci), the natural insect vector of the virus, and subjected to RFLP analysis. Markers on chromosomes 3 and 6 were significantly associated with the level of tolerance; the association of chromosome-6 markers was further substantiated in two additional BC2S1 populations. A tolerant BC2S1 plant which was homozygous for L. chilense introgressions in chromosomes 3, 6 and 7 was crossed to generate a BC3S1 population which was planted in an infested field. A TYLCV-tolerance gene with partial dominance, TY-1, was mapped to chromosome 6; two modifier genes were mapped to chromosomes 3 and 7. Field and whitefly-mediated cage inoculations of nearly-isogenic lines in BC3S3 supported our conclusion that TY-1 is the major TYLCV-tolerance locus.
We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F2 population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.
The inheritance of resistance to powdery mildew (Oidium lycopersicum) in Lycopersicon hirsutum was investigated by disease tests in segregating populations obtained by hybridising tomato (L. esculentum) cv Moneymaker with the wild relative L. hirsutum G1.1560. One incompletely dominant gene Ol-1 was found to largely control resistance to the disease. To map Ol-1, DNA pools from seven resistant and ten susceptible F2 plants were analyzed for random amplified polymorphic DNA (RAPD). With 32 primers tested, one RAPD, primed with the sequence 5'-GACGTGGTGA-3', was observed between the susceptible and the resistant bulks, which cosegregated with resistance in the F2 population of L. esculentum × L. hirsutum G1.1560. This RAPD was mapped on chromosome 6 by using an F2 (L. esculentum × L. pennellii) already mapped for 49 RFLPs. RFLP analysis of the F2 from L. esculentum cv Moneymaker × L. hirsutum G1.1560 demonstrated that Ol-1 maps near the Aps-1 region on chromosome 6, in the vicinity of the resistance genes to Meloidogyne spp. (Mi) and to Cladosporium fulvum (Cf-2/Cf-5).
In potato, 11 resistance alleles (R1-R11) are known which confer race-specific resistance to the fungus Phytophthora infestans. R1 has been mapped previously to potato chromosome V and R3 to chromosome XI. Here we report on the localization of the R6 and R7 alleles on the genetic map of potato. Differential resistant strains of tetraploid Solanum tuberosum, clones MaR6 and MaR7, were used as parental plants for the parthenogenetic induction and selection of diploid genotypes containing the R6 or the R7 resistance allele to P. infestans. One resistant dihaploid from MaR7 could be used directly as a parent to produce diploid F1 progeny suitable for phenotypic and RFLP analysis. MaR6 did not produce useful dihaploids directly. After crossing MaR6 with a tetraploid susceptible genotype, resistant F1 clones were selected. The resistant genotypes were then used as parents for the induction of dihaploids. Six dihaploids bearing R6 were identified that could be crossed with a diploid susceptible genotype. Two diploid F1 populations, segregating for R6 and R7, respectively, were analysed with RFLP markers known to be linked with previously identified R genes. Markers linked with R3 were found also to be linked with R6 and R7. The resistance alleles R6 and R7 mapped to a similar distal position on chromosome XI as the R3 allele.
Four different populations segregating for one of the two closely linked (possibly allelic) tomato disease resistance genes to the fungusCladosporium fulvum,Cf-4 andCf-9, were generated and analysed for recombination frequencies between theCf-genes and restriction fragment length polymorphism (RFLP) loci. The population consisting of F2 progeny from the interspecific crossLycopersicon esculentum carryingCf-9 ×L. pennellii was identified as the most useful for RFLP mapping of theCf-4/9 locus and an RFLP map around this locus was constructed mainly using this population. The two closest markers identified were CP46, 2.6 cM distal, and a group of 11 markers including TG236, 3.7 cM proximal toCf-4/9. A polymerase chain reaction (PCR)-based procedure for the rapid identification of recombination events between these two markers was developed. The regions of foreign DNA introgression surroundingCf-4 andCf-9 in near-isogenic lines were delimited.
In plants, resistance to pathogens is frequently determined by dominant resistance genes, whose products are proposed to recognize pathogen-encoded avirulence gene (Avr) products. The tomato resistance locus Cf-2 was isolated by positional cloning and found to contain two almost identical genes, each conferring resistance to isolates of tomato leaf mould (C. fulvum) expressing the corresponding Avr2 gene. The two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat (LRR) motifs. Of the LRRs, 20 show extremely conserved alternating repeats. The C-terminus of Cf-2 carries regions of pronounced homology to the protein encoded by the unlinked Cf-9 gene. We suggest that this conserved region interacts with other proteins involved in activating plant defense mechanisms.
The chromosomal location of the major gene Ry
adg
controlling extreme resistance to potato virus Y (PVY) in Solanum tuberosum subsp. andigena was identified by RFLP analysis of a diploid potato population. A total of 64 tomato and potato RFLP markers were screened with the bulked segregant analysis (BSA) on segregants extremely resistant, hypersensitive or susceptible to PVY. Four markers TG508, GP125, CD17 and CT168 at the proximal end of chromosome XI showed close linkage with extremely resistant phenotypes. TG508 was identified as the closest marker linked with the Ry
adg
locus with the maximum map distance estimated as 2.0 cM. The 4 markers linked with the Ry
adg
locus were tested on independent tetraploid and diploid potato clones and were subsequently found useful for marker-assisted selection for plants containing Ry
adg
.
The dominant gene I2 confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I2. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I2 locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F2 plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centiMorgan (CM) from I2. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I2. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I2 by chromosome walking from TG105.
Late blight in potato is caused by the fungus Phytophthora infestans and can inflict severe damage on the potato crop. Resistance to P. infestans is either based on major dominant R genes conferring vertical, race-specific resistance or on "minor" genes inducing horizontal, unspecific resistance. A dihaploid potato line was identified which carried the R1 gene, conferring vertical resistance to all P. infestans races, with the exception of those homozygous for the recessive virulence allele of the locus V1. The F1 progeny of a cross between this resistant parent P(R1) and P(r), a line susceptible to all races, was analysed for segregation of R1 and of restriction fragment length polymorphism (RFLP) markers distributed on the potato RFLP map comprising more than 300 loci. The R1 locus was mapped to chromosome V in the interval between RFLP markers GP21 and GP179. The map position of R1 was found to be very similar to the one of Rx2, a dominant locus inducing extreme resistance to potato virus X.
The HM1 gene in maize controls both race-specific resistance to the fungus Cochliobolus carbonum race 1 and expression of
the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-dependent HC toxin reductase (HCTR), which inactivates
HC toxin, a cyclic tetrapeptide produced by the fungus to permit infection. Several HM1 alleles were generated and cloned
by transposon-induced mutagenesis. The sequence of wild-type HM1 shares homology with dihydroflavonol-4-reductase genes from
maize, petunia, and snap-dragon. Sequence homology is greatest in the beta alpha beta-dinucleotide binding fold that is conserved
among NADPH- and NADH (reduced form of nicotinamide adenine dinucleotide)-dependent reductases and dehydrogenases. This indicates
that HM1 encodes HCTR.
Two different chromosomal locations of major genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length polymorphism (RFLP) analysis of two populations segregating for the resistance. The resistance gene Rx1 mapped to the distal end of chromosome XII, whereas Rx2 was located at an intermediate position on linkage group V in a region where reduced recombination and segregation distortion have also been observed. These linkage anomalies were due to abnormal behaviour of the chromosome contributed by the resistant parent P34. The results presented were obtained using two different strategies for mapping genes of unknown location. One approach was the use of probes revealing polymorphic loci spread throughout the genome and resulted in the mapping of Rx1. The second approach was based on the assumption of possible linkage between the resistance gene and clone-specific DNA fragments introduced from a wild potato species. Rx2 was mapped by adopting this strategy.
The construction of a restriction fragment length polymorphism (RFLP) linkage map is based on the estimation of recombination frequencies between genetic loci and on the determination of the linear order of loci in linkage groups. RFLP loci can be identified as segregations of singular or allelic DNA-restriction fragments. From crosses between heterozygous individuals several allele (fragment) configurations are possible, and this leads to a set of formulas for the evaluation of p, the recombination frequency between two loci. Tables and figures are presented illustrating a general outline of gene mapping using heterozygous populations. The method encompasses as special cases the mapping of loci from segregating populations of pure lines. Formulas for deriving the recombination frequencies and information functions are given for different fragment configurations. Information functions derived for relevant configurations are also compared. A procedure for map construction is presented, as it has been applied to RFLP mapping in an allogamous crop.
Many ATP- and GTP-binding proteins have a phosphate-binding loop (P-loop), the primary structure of which typically consists of a glycine-rich sequence followed by a conserved lysine and a serine or threonine. The three-dimensional structures of several ATP- and GTP-binding proteins containing P-loops have now been solved. In this review current knowledge of P-loops is discussed with the additional aim of illustrating the fascinating relationship between protein sequence, structure and function.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
The L6 rust resistance gene from flax was cloned after tagging with the maize transposable element Activator. The gene is predicted to encode two products of 1294 and 705 amino acids that result from alternatively spliced transcripts. The longer product is similar to the products of two other plant disease resistance genes, the tobacco mosaic virus resistance gene N of tobacco and the bacterial resistance gene RPS2 of Arabidopsis. The similarity involves the presence of a nucleotide (ATP/GTP) binding site and several other amino acid motifs of unknown function in the N-terminal half of the polypeptides and a leucine-rich region in the C-terminal half. The truncated product of L6, which lacks most of the leucine-rich C-terminal region, is similar to the truncated product that is predicted from an alternative transcript of the N gene. The L6, N, and RPS2 genes, which control resistance to three widely different pathogen types, are the foundation of a class of plant disease resistance genes that can be referred to as nucleotide binding site/leucine-rich repeat resistance genes.
In a random sample of 2263 cloned genomic DNA fragments of the wild potato species Solanum spegazzinii six related, highly repetitive fragments (SPG repeat family) were identified that were present in much higher copy numbers in S. spegazzinii when compared with the closely related cultivated potato S. tuberosum. The SPG repeat family was organized in long arrays of multiple copies. Cross hybridization experiments with 29 wild and cultivated Solanum species and with the related tomato showed specificity of the SPG repeat family for tuber-bearing Solanum species. Among tuber bearing Solanum species a high degree of variation was observed for restriction fragment length and copy number. The variation in copy number was not correlated with established taxonomic relationships between tuber-bearing Solanum species. DNA sequence analysis revealed a subrepeat structure of 120-140 base pairs embedded in longer repeat units of variable length. Length polymorphisms between highly repeated restriction fragments detected by the SPG probes were used for segregation- and linkage analysis in four mapping populations of potato, for which RFLP maps had been constructed. Twelve loci were identified, eleven of which mapped to the distal ends of nine linkage groups. All the evidence suggested that the SPG repeat family represents a satellite repeat members of which are localized in the subtelomeric region of potato chromosomes. The SPG repeat family could be used, therefore, for completing the genetic map of potato.
The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.
During the adaptation of plants to low temperature, changes in gene expression can be induced in a variety of tissues. Low-temperature-regulated gene expression was studied in cold-stored potato (Solanum tuberosum L.) tubers by two-dimensional electrophoresis of in vitro translation products. As a response to cold treatment, the relative amount of mRNA encoding at least 26 polypeptides changed. By differential screening of a cDNA library, 16 clones corresponding to cold-inducible transcripts were isolated. They were classified into four non-cross-hybridizing groups. RNA hybridizations using representative clones from each group revealed different temporal accumulation patterns for the cold-inducible transcripts. mRNAs homologous to the cDNA clones were first detectable after 1 to 3 d of cold treatment, and the highest level of expression was reached after 3 to 7 d. Transcripts corresponding to cDNA clones CI13 and CI19 were transiently expressed, whereas the steady-state level remained high for cDNA clones CI7 and CI21 during the cold storage period of 4 weeks. The DNA sequences of two cDNA clones, CI7 and CI19, have been determined. The polypeptide predicted from the DNA sequence of CI19 is sequence related to small heat-shock proteins from other plant species. The deduced protein sequence of CI7 exhibits strong homology to the dehydrin/RAB group of dehydration stress- and abscisic acid-inducible polypeptides and to cold-induced proteins from Arabidopsis and spinach.
Phytophthora infestans is the most important fungal pathogen in the cultivated potato (Solanum tuberosum). Dominant, race-specific resistance alleles and quantitative resistance--the latter being more important for potato breeding--are found in the germplasm of cultivated and wild potato species. Quantitative trait loci (QTLs) for resistance to two races of P. infestans have been mapped in an F1 progeny of a cross between non-inbred diploid potato parents with multiple alleles. Interval mapping methods based on highly informative restriction fragment length polymorphism markers revealed 11 chromosome segments on 9 potato chromosomes showing significant contrasts between marker genotypic classes. Whereas phenotypically no difference in quantitative resistance response was observed between the two fungal races, QTL mapping identified at least one race specific QT locus. Two QT regions coincided with two small segments on chromosomes V and XII to which the dominant alleles R1, conferring race specific resistance to P. infestans, Rx1 and Rx2, both inducing extreme resistance to potato virus X, have been allocated in independent mapping experiments. Some minor QTLs were correlated with genetic loci for specific proteins related to pathogenesis, the expression of which is induced after infection with P. infestans.
In plants, resistance to a pathogen is frequently correlated with a genetically defined interaction between a plant resistance gene and a corresponding pathogen avirulence gene. A simple model explains these gene-for-gene interactions: avirulence gene products generate signals (ligands), and resistance genes encode cognate receptors. The A. thaliana RPS2 gene confers resistance to the bacterial pathogen P. syringae carrying the avirulence gene avrRpt2. A map-based positional cloning strategy was used to identify RPS2. The identification of RPS2 was verified using a newly developed transient assay for RPS2 function and by genetic complementation in transgenic plants. RPS2 encodes a novel 105 kDa protein containing a leucine zipper, a nucleotide-binding site, and 14 imperfect leucine-rich repeats.