RESUMO
BACKGROUND: Scaffolding is an essential step in the genome assembly process. Current methods based on large fragment paired-end reads or long reads allow an increase in contiguity but often lack consistency in repetitive regions, resulting in fragmented assemblies. Here, we describe a novel tool to link assemblies to a genome map to aid complex genome reconstruction by detecting assembly errors and allowing scaffold ordering and anchoring. RESULTS: We present MaGuS (map-guided scaffolding), a modular tool that uses a draft genome assembly, a Whole Genome Profiling™ (WGP) map, and high-throughput paired-end sequencing data to estimate the quality and to enhance the contiguity of an assembly. We generated several assemblies of the Arabidopsis genome using different scaffolding programs and applied MaGuS to select the best assembly using quality metrics. Then, we used MaGuS to perform map-guided scaffolding to increase contiguity by creating new scaffold links in low-covered and highly repetitive regions where other commonly used scaffolding methods lack consistency. CONCLUSIONS: MaGuS is a powerful reference-free evaluator of assembly quality and a WGP map-guided scaffolder that is freely available at https://github.com/institut-de-genomique/MaGuS. Its use can be extended to other high-throughput sequencing data (e.g., long-read data) and also to other map data (e.g., genetic maps) to improve the quality and the contiguity of large and complex genome assemblies.
Assuntos
Arabidopsis/genética , Cromossomos de Plantas/genética , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mapeamento Físico do Cromossomo , Análise de Sequência de DNA/métodos , Cromossomos Artificiais Bacterianos , Mapeamento de Sequências Contíguas , Sequências Repetitivas de Ácido Nucleico , Alinhamento de SequênciaRESUMO
Late blight caused by the plant pathogenic oomycete Phytophthora infestans is known as one of the most destructive potato diseases. Plant breeders tend to employ NB-LRR-based resistance for introducing genetically controlled late blight resistance in their breeding lines. However, P. infestans is able to rapidly escape this type of resistance, and hence, NB-LRR-based resistance in potato cultivars is often not durable. Previously, we identified a novel type of Phytophthora resistance in Arabidopsis. This resistance is mediated by the cell surface receptor LecRK-I.9, which belongs to the family of L-type lectin receptor kinases. In this study, we report that expression of the Arabidopsis LecRK-I.9 gene in potato and Nicotiana benthamiana results in significantly enhanced late blight resistance. Transcriptional profiling showed strong reduction in salicylic acid (SA)-mediated defence gene expression in LecRK-I.9 transgenic potato lines (TPLs). In contrast, transcripts of two protease inhibitor genes accumulated to extreme high levels, suggesting that LecRK-I.9-mediated late blight resistance is relying on a defence response that includes activation of protease inhibitors. These results demonstrate that the functionality of LecRK-I.9 in Phytophthora resistance is maintained after interfamily transfer to potato and N. benthamiana and suggest that this novel type of LecRK-based resistance can be exploited in breeding strategies to improve durable late blight resistance in Solanaceous crops.
Assuntos
Arabidopsis/metabolismo , Phytophthora infestans/patogenicidade , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/metabolismo , Solanum tuberosum/parasitologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Solanum tuberosum/genéticaRESUMO
Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring 'nontoxic' provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F2 mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F2 plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker-assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties.
Assuntos
Ligação Genética , Jatropha/genética , Ésteres de Forbol/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas , Marcadores Genéticos , Jatropha/metabolismo , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Sementes/genética , Sementes/metabolismoRESUMO
Marker development for marker-assisted selection in plant breeding is increasingly based on next-generation sequencing (NGS). However, marker development in crops with highly repetitive, complex genomes is still challenging. Here we applied sequence-based genotyping (SBG), which couples AFLP®-based complexity reduction to NGS, for de novo single nucleotide polymorphisms (SNP) marker discovery in and genotyping of a biparental durum wheat population. We identified 9983 putative SNPs in 6372 contigs between the two parents and used these SNPs for genotyping 91 recombinant inbred lines (RILs). Excluding redundant information from multiple SNPs per contig, 2606 (41%) markers were used for integration in a pre-existing framework map, resulting in the integration of 2365 markers over 2607 cM. Of the 2606 markers available for mapping, 91% were integrated in the pre-existing map, containing 708 SSRs, DArT markers, and SNPs from CRoPS technology, with a map-size increase of 492 cM (23%). These results demonstrate the high quality of the discovered SNP markers. With this methodology, it was possible to saturate the map at a final marker density of 0.8 cM/marker. Looking at the binned marker distribution (Figure 2), 63 of the 268 10-cM bins contained only SBG markers, showing that these markers are filling in gaps in the framework map. As to the markers that could not be used for mapping, the main reason was the low sequencing coverage used for genotyping. We conclude that SBG is a valuable tool for efficient, high-throughput and high-quality marker discovery and genotyping for complex genomes such as that of durum wheat.
Assuntos
Técnicas de Genotipagem , Polimorfismo de Nucleotídeo Único , Triticum/genética , Produtos Agrícolas/genética , Marcadores Genéticos , Genoma de PlantaRESUMO
Research on diploid hybrid potato has made fast advances in recent years. In this review we give an overview of the most recent and relevant research outcomes. We define different components needed for a complete hybrid program: inbred line development, hybrid evaluation, cropping systems and variety registration. For each of these components the important research results are discussed and the outcomes and issues that merit further study are identified. We connect fundamental and applied research to application in a breeding program, based on the experiences at the breeding company Solynta. In the concluding remarks, we set hybrid breeding in a societal perspective, and we identify bottlenecks that need to be overcome to allow successful adoption of hybrid potato.
RESUMO
BACKGROUND: Sequencing projects using a clone-by-clone approach require the availability of a robust physical map. The SNaPshot technology, based on pair-wise comparisons of restriction fragments sizes, has been used recently to build the first physical map of a wheat chromosome and to complete the maize physical map. However, restriction fragments sizes shared randomly between two non-overlapping BACs often lead to chimerical contigs and mis-assembled BACs in such large and repetitive genomes. Whole Genome Profiling (WGP™) was developed recently as a new sequence-based physical mapping technology and has the potential to limit this problem. RESULTS: A subset of the wheat 3B chromosome BAC library covering 230 Mb was used to establish a WGP physical map and to compare it to a map obtained with the SNaPshot technology. We first adapted the WGP-based assembly methodology to cope with the complexity of the wheat genome. Then, the results showed that the WGP map covers the same length than the SNaPshot map but with 30% less contigs and, more importantly with 3.5 times less mis-assembled BACs. Finally, we evaluated the benefit of integrating WGP tags in different sequence assemblies obtained after Roche/454 sequencing of BAC pools. We showed that while WGP tag integration improves assemblies performed with unpaired reads and with paired-end reads at low coverage, it does not significantly improve sequence assemblies performed at high coverage (25x) with paired-end reads. CONCLUSIONS: Our results demonstrate that, with a suitable assembly methodology, WGP builds more robust physical maps than the SNaPshot technology in wheat and that WGP can be adapted to any genome. Moreover, WGP tag integration in sequence assemblies improves low quality assembly. However, to achieve a high quality draft sequence assembly, a sequencing depth of 25x paired-end reads is required, at which point WGP tag integration does not provide additional scaffolding value. Finally, we suggest that WGP tags can support the efficient sequencing of BAC pools by enabling reliable assignment of sequence scaffolds to their BAC of origin, a feature that is of great interest when using BAC pooling strategies to reduce the cost of sequencing large genomes.
Assuntos
Genoma de Planta , Mapeamento Físico do Cromossomo , Análise de Sequência de DNA/métodos , Triticum/genética , Cromossomos Artificiais Bacterianos , Cromossomos de Plantas , Mapeamento de Sequências Contíguas , Elementos de DNA Transponíveis , Alinhamento de SequênciaRESUMO
The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD36(45-1), suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34-amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2.
Assuntos
Proteínas de Algas/metabolismo , Phytophthora infestans/patogenicidade , Proteínas de Plantas/metabolismo , Solanum/genética , Alelos , Sequência de Aminoácidos , Morte Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Imunidade Inata , Dados de Sequência Molecular , Phytophthora infestans/metabolismo , Proteínas de Plantas/genética , Polimorfismo Genético , RNA de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Solanum/imunologia , Solanum/metabolismo , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/metabolismoRESUMO
Late blight caused by Phytophthora infestans is the most important disease of potato. Many efforts have been made to understand molecular mechanism of the durable resistance to address the challenge raised by rapid evolution of the pathogen. A pathogenesis related protein (PR) gene StPRp27 was previously isolated from the potato leaves challenged by P. infestans. The sequence analysis and expression pattern reveal that StPRp27 may be associated with resistance to P. infestans. In present research, transient expression of StPRp27 in Nicotiana benthamiana enhanced resistance to P. infestans isolates 99189 and PY23 indicating its potential contribution to the disease resistance. These findings were also confirmed by over-expression of StPRp27 in potato cv. E-potato 3, which significantly slowed down the development of the disease after inoculation with a mixture of P. infestans races. Further, silencing of StPRp27 homologous genes in N. benthamiana harboring dominant Phytophthora resistance gene Rpi-blb1 or Rpi-blb2 showed no effects on the resistance triggered by these R genes. Our results suggest that StPRp27 contributes to a race-nonspecific resistance against P. infestans by inhibiting the disease development and has a potential use in selection and breeding for durable resistance to late blight.
Assuntos
Resistência à Doença/genética , Phytophthora infestans , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Solanum tuberosum , Cruzamento/métodos , Primers do DNA/genética , Inativação Gênica , Reação em Cadeia da Polimerase em Tempo Real , NicotianaRESUMO
Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.
Assuntos
Genes de Plantas , Phytophthora infestans/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Agrobacterium tumefaciens/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Clonagem Molecular , DNA de Plantas/genética , Teste de Complementação Genética , Interações Hospedeiro-Patógeno/genética , Família Multigênica , Filogenia , Doenças das Plantas/prevenção & controle , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Nicotiana/genética , Nicotiana/microbiologia , Transformação Genética , VirulênciaRESUMO
We describe the application of complexity reduction of polymorphic sequences (CRoPS(®)) technology for the discovery of SNP markers in tetraploid durum wheat (Triticum durum Desf.). A next-generation sequencing experiment was carried out on reduced representation libraries obtained from four durum cultivars. SNP validation and minor allele frequency (MAF) estimate were carried out on a panel of 12 cultivars, and the feasibility of genotyping these SNPs in segregating populations was tested using the Illumina Golden Gate (GG) technology. A total of 2,659 SNPs were identified on 1,206 consensus sequences. Among the 768 SNPs that were chosen irrespective of their genomic repetitiveness level and assayed on the Illumina BeadExpress genotyping system, 275 (35.8%) SNPs matched the expected genotypes observed in the SNP discovery phase. MAF data indicated that the overall SNP informativeness was high: a total of 196 (71.3%) SNPs had MAF >0.2, of which 76 (27.6%) showed MAF >0.4. Of these SNPs, 157 were mapped in one of two mapping populations (Meridiano × Claudio and Colosseo × Lloyd) and integrated into a common genetic map. Despite the relatively low genotyping efficiency of the GG assay, the validated CRoPS-derived SNPs showed valuable features for genomics and breeding applications such as a uniform distribution across the wheat genome, a prevailing single-locus codominant nature and a high polymorphism. Here, we report a new set of 275 highly robust genome-wide Triticum SNPs that are readily available for breeding purposes.
Assuntos
Genoma de Planta , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único , Triticum/genética , Cruzamento , Mapeamento Cromossômico , Cruzamentos Genéticos , DNA de Plantas/genética , Frequência do Gene , Marcadores Genéticos , Técnicas de Genotipagem , Análise de Sequência de DNA , TetraploidiaRESUMO
The H1 locus confers resistance to the potato cyst nematode Globodera rostochiensis pathotypes 1 and 4. It is positioned at the distal end of chromosome V of the diploid Solanum tuberosum genotype SH83-92-488 (SH) on an introgression segment derived from S. tuberosum ssp. andigena. Markers from a high-resolution genetic map of the H1 locus (Bakker et al. in Theor Appl Genet 109:146-152, 2004) were used to screen a BAC library to construct a physical map covering a 341-kb region of the resistant haplotype coming from SH. For comparison, physical maps were also generated of the two haplotypes from the diploid susceptible genotype RH89-039-16 (S. tuberosum ssp. tuberosum/S. phureja), spanning syntenic regions of 700 and 319 kb. Gene predictions on the genomic segments resulted in the identification of a large cluster consisting of variable numbers of the CC-NB-LRR type of R genes for each haplotype. Furthermore, the regions were interspersed with numerous transposable elements and genes coding for an extensin-like protein and an amino acid transporter. Comparative analysis revealed a major lack of gene order conservation in the sequences of the three closely related haplotypes. Our data provide insight in the evolutionary mechanisms shaping the H1 locus and will facilitate the map-based cloning of the H1 resistance gene.
Assuntos
Loci Gênicos/genética , Haplótipos/genética , Imunidade Inata/genética , Doenças das Plantas/imunologia , Análise de Sequência de DNA , Solanum tuberosum/genética , Solanum tuberosum/parasitologia , Cromossomos de Plantas/genética , Elementos de DNA Transponíveis/genética , Genoma de Planta/genética , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/imunologiaRESUMO
Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato.
Assuntos
Mapeamento Cromossômico/métodos , Doenças das Plantas/genética , Locos de Características Quantitativas , Solanum tuberosum/genética , Clonagem Molecular , Resistência à Doença , Perfilação da Expressão Gênica , Biblioteca Gênica , Genes de Plantas , Ligação Genética , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteínas de Plantas/química , Proteínas de Plantas/genética , Análise de Sequência de DNA , Solanum tuberosum/imunologiaRESUMO
Knowledge on the evolution and distribution of late blight resistance genes is important for a better understanding of the dynamics of these genes in nature. We analyzed the presence and allelic diversity of the late blight resistance genes Rpi-blb1, Rpi-blb2, and Rpi-blb3, originating from Solanum bulbocastanum, in a set of tuber-bearing Solanum species comprising 196 different taxa. The three genes were only present in some Mexican diploid as well as polyploid species closely related to S. bulbocastanum. Sequence analysis of the fragments obtained from the Rpi-blb1 and Rpi-blb3 genes suggests an evolution through recombinations and point mutations. For Rpi-blb2, only sequences identical to the cloned gene were found in S. bulbocastanum accessions, suggesting that it has emerged recently. The three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their spread is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2, and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2, and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach, some accessions could be identified that contain novel, as yet unknown, late blight resistance factors in addition to the Rpi-blb1, Rpi-blb2, and Rpi-blb3 genes.
Assuntos
Evolução Biológica , Doenças das Plantas/genética , Solanum/microbiologia , DNA de Plantas , Variação Genética , Doenças das Plantas/imunologia , Reação em Cadeia da PolimeraseRESUMO
Despite the efforts of breeders and the extensive use of fungicide control measures, late blight still remains a major threat to potato cultivation worldwide. The introduction of genetic resistance into cultivated potato is considered a valuable method to achieve durable resistance to late blight. Here, we report the identification and cloning of Rpi-vnt1.1, a previously uncharacterized late-blight resistance gene from Solanum venturii. The gene was identified by a classical genetic and physical mapping approach and encodes a coiled-coil nucleotide-binding leucine-rich repeat protein with high similarity to Tm-2(2) from S. lycopersicum which confers resistance against Tomato mosaic virus. Transgenic potato and tomato plants carrying Rpi-vnt1.1 were shown to be resistant to Phytophthora infestans. Of 11 P. infestans isolates tested, only isolate EC1 from Ecuador was able to overcome Rpi-vnt1.1 and cause disease on the inoculated plants. Alleles of Rpi-vnt1.1 (Rpi-vnt1.2 and Rpi-vnt1.3) that differed by only a few nucleotides were found in other late-blight-resistant accessions of S. venturii. The late blight resistance gene Rpi-phu1 from S. phureja is shown here to be identical to Rpi-vnt1.1, suggesting either that this strong resistance gene has been maintained since a common ancestor, due to selection pressure for blight resistance, or that genetic exchange between S. venturii and S. phureja has occurred at some time.
Assuntos
Genes de Plantas/genética , Doenças das Plantas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Solanum/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Clonagem Molecular , DNA de Plantas/química , DNA de Plantas/genética , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Dados de Sequência Molecular , Phytophthora infestans/fisiologia , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanum/classificação , Solanum/microbiologia , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Especificidade da EspécieRESUMO
Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases of potato. Resistance (R) genes from the wild species Solanum demissum have been used by breeders to generate late-blight-resistant cultivars but resistance was soon overcome by the pathogen. A more recent screening of a large number of wild species has led to the identification of novel sources of resistance, many of which are currently being characterized further. Here, we report on the cloning of dominant Rpi genes from S. venturii. Rpi-vnt1.1 and Rpi-vnt1.3 were mapped to chromosome 9 using nucleotide binding site (NBS) profiling. Subsequently, a Tm-2(2)-based allele mining strategy was used to clone both genes. Rpi-vnt1.1 and Rpi-vnt1.3 belong to the coiled-coil NBS leucine-rich repeat (LRR) class of plant R genes and encode predicted peptides of 891 and 905 amino acids (aa), respectively, which share 75% amino acid identity with the Tomato mosaic virus resistance protein Tm-2(2) from tomato. Compared with Rpi-vnt1.1, Rpi-vnt1.3 harbors a 14-aa insertion in the N-terminal region of the protein and two different amino acids in the LRR domain. Despite these differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , Doenças das Plantas/genética , Solanum/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA de Plantas/química , DNA de Plantas/genética , Teste de Complementação Genética , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Dados de Sequência Molecular , Phytophthora infestans/fisiologia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanum/crescimento & desenvolvimento , Solanum/microbiologia , Nicotiana/genéticaRESUMO
A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1-mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1-mediated resistance in potato.
Assuntos
Proteínas Fúngicas/metabolismo , Phytophthora infestans/genética , Phytophthora infestans/metabolismo , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Variação Genética , Dados de Sequência Molecular , Filogenia , Phytophthora infestans/patogenicidade , VirulênciaRESUMO
In addition to the resistance to Phytophthora infestans (Rpi) genes Rpi-blb1 and Rpi-blb2, Solanum bulbocastanum appears to harbor Rpi-blb3 located at a major late blight resistance locus on LG IV, which also harbors Rpi-abpt, R2, R2-like, and Rpi-mcd1 in other Solanum spp. Here, we report the cloning and functional analyses of four Rpi genes, using a map-based cloning approach, allele-mining strategy, Gateway technology, and transient complementation assays in Nicotiana benthamiana. Rpi-blb3, Rpi-abpt, R2, and R2-like contain all signature sequences characteristic of leucine zipper nucleotide binding site leucine-rich repeat (LZ-NBS-LRR) proteins, and share amino-acid sequences 34.9% similar to RPP13 from Arabidopsis thaliana. The LRR domains of all four Rpi proteins are highly homologous whereas LZ and NBS domains are more polymorphic, those of R2 being the most divergent. Clear blocks of sequence affiliation between the four functional resistance proteins and those encoded by additional Rpi-blb3 gene homologs suggest exchange of LZ, NBS, and LRR domains, underlining the modular nature of these proteins. All four Rpi genes recognize the recently identified RXLR effector PiAVR2.
Assuntos
Phytophthora infestans , Doenças das Plantas/genética , Proteínas de Plantas/genética , Solanum tuberosum/genética , Alelos , Sequência de Aminoácidos , Clonagem Molecular , Teste de Complementação Genética , Imunidade Inata/genética , Dados de Sequência Molecular , Família Multigênica , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/microbiologia , Alinhamento de Sequência , Nicotiana/genéticaRESUMO
Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC-FISH as a unique tool for comparative genetic studies across Solanum species.
Assuntos
Aberrações Cromossômicas , Cromossomos Artificiais Bacterianos , Cromossomos de Plantas/genética , Hibridização in Situ Fluorescente/métodos , Mapeamento Físico do Cromossomo/métodos , Solanum lycopersicum/genética , Solanum tuberosum/genética , DNA de Plantas/genética , Marcadores Genéticos , Genoma de Planta , Especificidade da EspécieRESUMO
Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (â¼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.
RESUMO
Sugarcane (Saccharum spp.) is a major crop for sugar and bioenergy production. Its highly polyploid, aneuploid, heterozygous, and interspecific genome poses major challenges for producing a reference sequence. We exploited colinearity with sorghum to produce a BAC-based monoploid genome sequence of sugarcane. A minimum tiling path of 4660 sugarcane BAC that best covers the gene-rich part of the sorghum genome was selected based on whole-genome profiling, sequenced, and assembled in a 382-Mb single tiling path of a high-quality sequence. A total of 25,316 protein-coding gene models are predicted, 17% of which display no colinearity with their sorghum orthologs. We show that the two species, S. officinarum and S. spontaneum, involved in modern cultivars differ by their transposable elements and by a few large chromosomal rearrangements, explaining their distinct genome size and distinct basic chromosome numbers while also suggesting that polyploidization arose in both lineages after their divergence.